JP2017014239A - Modified single domain antigen binding molecules and uses thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide methods of preparing modified single domain antigen-binding molecules for treating TNFa-related disorders and to provide methods of using the same, the methods relating to modified single domain antigen-binding molecules (SDAB molecules), particularly relating to TNFa-binding SDAB molecules.SOLUTION: The present invention provides a modified single domain antigen binding molecule comprising (i) one or more single antigen binding domains that bind to one or more targets (e.g. TNFa); (ii) a non-peptidic linker; and (iii) one or more polymer molecules (e.g. PEG), wherein for example, a linker and PEG polymer are represented by the following formula.SELECTED DRAWING: None

Description

  The present invention relates to modified single domain antigen binding molecules (also referred to herein as “SDAB molecules”).

  This application claims priority to US Provisional Application No. 61 / 365,307, filed July 16, 2010. The entire text of the above application is hereby incorporated by reference.

  Tumor necrosis factor alpha (TNFα) is a secreted and membrane-bound pro-inflammatory cytokine produced primarily by macrophages and monocytes. TNFα synthesis is upregulated in various chronic autoimmune inflammatory diseases such as rheumatoid arthritis, ulcerative colitis, Crohn's disease and the like. TNFα is expressed as a trimeric transmembrane protein that can be cleaved by TNFα converting enzyme (TACE) and release its soluble form. Both types of TNFα interact with TNF receptor (TNFR) 1 and TNFR2.

  A modified SDAB molecule can include one or more single antigen binding domains that interact with one or more targets, eg, bind to one or more targets. In one embodiment, one or more of the single antigen binding domains of the modified SDAB molecule binds to tumor necrosis factor-α (TNFα). An SDAB molecule can be modified to increase its biological properties in vivo. For example, an SDAB molecule has one or more of increased half-life, reduced immunogenicity, or improved at least one pharmacokinetic / pharmacodynamic (PK / PD) parameter compared to an unmodified SDAB molecule. It can be modified to improve. In one embodiment, the modified SDAB molecule includes one or more polymer molecules, such as poly (ethylene glycol) (PEG) or derivatives thereof. Modified SDAB molecules are useful, for example, for administration to a subject, eg, a human. Also disclosed are methods of preparing and using, for example, modified SDAB molecules for treating or preventing TNFα-related disorders.

  Accordingly, in one aspect, the invention provides (i) one or more single antigen binding domains that interact with, eg, bind to, one or more targets (eg, TNFα), eg, characterized by a modified SDAB molecule comprising a linker (eg a non-peptide linker and / or a peptide linker) and (iii) one or more polymer molecules, eg poly (ethylene glycol) (PEG) or derivatives thereof To do. In one embodiment, the SDAB molecule linker is a non-peptide linker. In certain embodiments, the SDAB molecule can be modified by, for example, covalently or non-covalently associated with a second moiety, eg, a polymer molecule. For example, the SDAB molecule can be covalently attached to a suitable pharmacologically acceptable polymer, such as poly (ethylene glycol) (PEG) or a derivative thereof (eg methoxy poly (ethylene glycol) or mPEG).

  In one embodiment, the modified SDAB molecule comprises one or more single binding domains. For example, the SDAB molecule may comprise a polypeptide comprising at least one immunoglobulin variable domain (including one, two, or three complementarity determining regions (CDRs)), such as a single chain polypeptide, Or it can consist of it. Examples of SDAB molecules include molecules that naturally lack a light chain (eg, VHH, Nanobodies, or camelid antibodies). Such SDAB molecules can be derived or obtained from camelids such as camels, llamas, dromedaries, alpaca and guanacos. In other embodiments, SDAB molecules include, but are not limited to, other naturally occurring single domain molecules (eg, shark single domain polypeptide (IgNAR)) and single domain scaffolds (eg, fibronectin scaffold). It may contain one or more single domain molecules that are not.

  In another embodiment, the modified SDAB molecule is a single chain polypeptide consisting of one or more single antigen binding domains. SDAB molecules can bind to the same target, eg, at the same or different epitope, or to different targets. The single antigen binding domain of the SDAB molecule can have the same or different amino acid sequence. In some embodiments, the SDAB molecule is monovalent or multivalent (eg, divalent, trivalent or tetravalent). In other embodiments, the SDAB molecule is monospecific or multispecific (eg, bispecific, trispecific or tetraspecific). SDAB molecules may contain one or more single antigen binding domains that are recombinant, CDR grafted, humanized, camelized, deimmunized, and / or generated in vitro (eg, selected by phage display). May be included. For example, an SDAB molecule can be a single chain fusion polypeptide comprising one, two, three, four or more single antigen binding domains that bind to one or more target antigens. Typically, the target antigen is a mammalian, eg, human protein. In one embodiment, the target antigen is TNFα, such as human TNFα.

  In one exemplary embodiment, the modified SDAB molecule is a single chain polypeptide fusion of two single antigen binding domains (eg, two camelid variable regions) that bind to a target antigen, eg, TNFα. It is a bivalent molecule consisting of The single antigen-binding domain of a modified SDAB molecule can be arranged from the N-terminus to the C-terminus in the following order: TNFα-linked single antigen-binding domain— (optionally a linking group such as a peptide linker) —TNFα Binding type single antigen-binding domain-one or more polymer molecules. In one embodiment, the single antigen binding domain binds to the same epitope on the target antigen (eg, using the same or different single antigen binding domains). In other embodiments, the single antigen binding domain of the SDAB molecule binds to different epitopes on the same or different targets. It will be understood that any order or combination of two, three, four or more single antigen binding domains for one or more targets is encompassed by the present invention.

In other embodiments, two, three, four or more of the single domain molecules of the modified SDAB molecule may be a gene fusion or polypeptide with or without a linking group. Associate as a fusion (eg, fuse). The linking group can be any linking group apparent to those of skill in the art. For example, the linking group can be a biocompatible polymer 1 to 100 atoms long. The linking group can be a peptide linker or a non-peptide linker. In one embodiment, the linking group is a peptide linker, for example, the linking group is a polyglycine residue, a polyserine residue, a polylysine residue, a polyglutamic acid residue, a polyisoleucine residue or a polyarginine residue, or Or a combination thereof. For example, the polyglycine linking group or polyserine linking group includes at least 5, 7, 8, 9, 10, 12, 15, 20, 30, 35 and 40 glycine residues. And serine residues may be included. Examples of linking groups that can be used include Gly-Ser repeats having at least 1, 2, 3, 4, 5, 6, 7, or more repeats, eg (Gly) ₃ -Ser (SEQ ID NO: 7) or (Gly) ₄ -Ser (SEQ ID NO: 8) repeats. In some embodiments, the linking group has the following sequence: (Gly) ₄ -Ser- (Gly) ₃ -Ser (SEQ ID NO: 9) or ((Gly) ₄ -Ser) n (SEQ ID NO: 10). (Where n is 4, 5 or 6). In one embodiment, the linking group comprises the following sequence: ((Gly) ₄ -Ser) n (SEQ ID NO: 10), where n = 6. A modified SDAB molecule further comprises a linking group (eg, referred to herein as a “C-terminal linking group”) at the C-terminus of a single antigen binding domain, and another moiety (eg, a carrier molecule, a non-peptide linker or The attachment of SDAB to (non-peptide moiety) can be facilitated. Any of the linking groups described herein can be used as a C-terminal linking group. In one embodiment, one or more Gly-Ser repeats are used, eg, one or more repeats of (Gly) ₃ -Ser or (Gly) ₄ -Ser (SEQ ID NO: 8).

  In one embodiment, the modified SDAB molecule (referred to herein as “SDAB-01”) has the amino acid sequence shown in FIG. 1 (SEQ ID NO: 1), or an amino acid substantially identical thereto. Sequence (eg, at least 85%, 90%, 95% or more identity or up to 20, 15, 10, 5, 4 compared to the amino acid sequence shown in FIG. 1) Comprise or consist of three, two, one amino acid changes (eg, amino acid sequences having deletions, insertions or substitutions (eg, conservative substitutions)). The nucleotide sequence encoding the two single antigen binding domains of SEQ ID NO: 1 is given as SEQ ID NO: 6 (see Table 12). In other embodiments, the modified SDAB is SEQ ID NO: 6, or a nucleotide sequence substantially identical thereto (eg, at least 85%, 90%, 95% or more compared to the amino acid sequence of SEQ ID NO: 6). Or an amino acid sequence encoded by a nucleotide sequence having a maximum of 60, 45, 30, 15, 12, 12, 9, 6, or 3 nucleotide changes) Or consist of.

  Examples of further single domain molecules include the amino acid sequences disclosed in Table 19 of WO 2006/122786 (which is hereby incorporated by reference) and Table 11 below. However, it is not limited to them.

  In certain embodiments, at least one of the single antigen binding domains of a modified SDAB molecule that binds to TNFα has the following amino acid sequence: DYWMY (SEQ ID NO: 2) (CDR1), EINTNGLITKYPDSVKG (SEQ ID NO: 3 ) (CDR2) and / or one, two or three CDRs with SPSGFN (SEQ ID NO: 4) (CDR3) or fewer than three, two or less amino acid substitutions (eg conservative substitutions) Has a CDR that differs from one of the CDRs described above. In other embodiments, the single antigen binding domain has an amino acid sequence of about amino acids 1-115 of FIG. 1 or an amino acid sequence substantially identical thereto (eg, at least 85 amino acids compared to the amino acid sequence shown in FIG. 1). %, 90%, 95% or more identity or up to 20, 15, 10, 5, 4, 3, 2, 1 amino acid changes (eg deletions) , Variable regions having amino acid sequences) with insertions or substitutions (eg conservative substitutions)). In some embodiments, the TNFα-binding SDAB molecule has one or more biological activities of the TNFα-binding single domain antibody molecule shown in FIG. For example, a TNFα-binding SDAB molecule binds to the same or similar epitope recognized by the TNFα-binding single domain molecule shown in FIG. 1 (eg, binds TNFα in its trimeric form; Binds to the TNFα site that contacts the TNF receptor; Gln at position 88 on the first TNF monomer (monomer A), Lys at position 90, and Glu at position 146 on the second TNF monomer (monomer B). Binds to an epitope in the TNFα trimer comprising, or disclosed in WO 2006/122786). In another embodiment, a TNFα-binding SDAB molecule binds to the N-terminus of TNFα. In other embodiments, the TNFα-binding SDAB molecule has similar activity (eg, binding affinity, dissociation constant, binding) to any of the TNFα-binding single domain molecules disclosed in WO 2006/122786. Specificity, TNFα inhibitory activity).

  In other embodiments, TNFα-binding SDAB molecules are disclosed in Table 11 and also disclosed in WO 2006/122786 (which is hereby incorporated by reference). Contains one or more. For example, the TNFα-binding SDAB molecule can be a monovalent, divalent, or trivalent TNFα-binding SDAB molecule disclosed in Table 9 of WO 2006/122786. Examples of TNFα-binding SDAB molecules include, but are not limited to, TNF1, TNF2, TNF3, and their humanized forms (eg, TNF29, TNF30, TNF31, TNF32, TNF33). Further examples of monovalent TNFα-binding SDAB molecules are disclosed in Table 8 of WO 2006/122786. An example of a bivalent TNFα-binding SDAB molecule includes two TNF30 SDAB molecules linked via a peptide linker to form a single fusion polypeptide (disclosed in WO 2006/122786) Include, but are not limited to, TNF55 and TNF56. Further examples of bivalent TNFα-binding SDAB molecules are disclosed as TNF4, TNF5, TNF6, TNF7, TNF8) in Table 11 of this specification or in Table 19 of WO 2006/122786.

  In certain embodiments, the SDAB molecule is modified to include one or more polymer molecules, such as poly (ethylene glycol) (PEG) or derivatives thereof. PEG molecules (eg, PEG monomers, polymers, or derivatives thereof) can be linear or branched. In one embodiment, SDAB is attached to one or more PEG molecules via a linker moiety (eg, a non-peptide linker).

In some embodiments, the linker is a non-peptide linker. In one embodiment, the linker is represented by formula (I):
(Where
W ¹ and W ² are each independently selected from a bond or NR ¹ and Y is a bond, C _1-4 alkylene substituted with 0-2 R ^a , or pyrrolidine-2,5-dione. ,
X is O, a bond or absent,
Z is absent or is O, NR ³ , S, or a bond,
R ¹ and R ³ are each independently hydrogen or C _1-6 alkyl;
R ² is absent or is one or more polymer moieties;
R ^a is selected from hydroxyl, C _1-4 alkyl or C _1-4 alkoxy;
m is 0 or 1,
n is 0, 1, 2 or 3;
p is 0, 1, 2, 3 or 4.

In some embodiments, one or more polymer moieties (eg, R ^{2 of} formula (I)) of the SDAB molecule are poly (ethylene glycol) (PEG) molecules (eg, PEG monomers, polymers, or derivatives thereof). including. In some embodiments, the PEG molecule is a methoxy poly (ethylene glycol) (mPEG) monomer, polymer, or derivative thereof.

In some embodiments, the PEG molecule is branched. In some embodiments, the PEG molecule is selected from moieties of formula (a) to formula (h):

(Where each PEG molecule is independently a PEG monomer, polymer, or derivative thereof). In some embodiments, each PEG molecule is an mPEG monomer, polymer, or derivative thereof.

In some embodiments, Y is a bond. In some embodiments, Y is pyrrolidine-2,5-dione. In some embodiments, Y is C _1-4 alkylene substituted with 0-2 R ^a . In some embodiments, Y is C _1-4 alkylene substituted with 1 R ^a . In some embodiments, Y is methylene substituted with 1 R ^a . In some embodiments, R ^a is hydroxyl.

  In some embodiments, X is a bond. In some embodiments, X is oxygen (O). In some embodiments, X is not present.

In some embodiments, R ² is (a).

In some embodiments, R ² is (g).

In some embodiments, W ¹ is a bond. In some embodiments, W ¹ is NR ¹ .

In some embodiments, W ² is a bond. In some embodiments, W ² is NR ¹ .

In some embodiments, R ¹ is hydrogen.

  In some embodiments, Z is O, S or a bond.

  In some embodiments, Z is O.

In some embodiments, R ³ is hydrogen.

  In some embodiments, m is 0. In some embodiments, m is 1.

  In some embodiments, n is 0. In some embodiments, n is 2. In some embodiments, n is 3.

  In some embodiments, p is 0. In some embodiments, p is 3.

  In some embodiments, each PEG molecule is independently a PEG monomer, polymer, or derivative thereof. In some embodiments, each PEG molecule is a methoxy PEG derivative (mPEG) monomer, polymer, or derivative thereof. In some embodiments, each PEG molecule independently has a molecular weight of 1 KDa to 100 KDa. In some embodiments, each PEG molecule independently has a molecular weight between 10 KDa and 50 KDa. In some embodiments, each PEG molecule independently has a molecular weight of 40 KDa. In some embodiments, each PEG molecule independently has a molecular weight of 15 KDa to 35 KDa. In some embodiments, each PEG molecule independently has a molecular weight of 30 KDa. In some embodiments, each PEG molecule independently has a molecular weight of 20 KDa. In some embodiments, each PEG molecule independently has a molecular weight of 17.5 KDa. In some embodiments, each PEG molecule independently has a molecular weight of 12.5 KDa. In some embodiments, each PEG molecule independently has a molecular weight of 10 KDa. In some embodiments, each PEG molecule has a molecular weight of 7.5 KDa. In some embodiments, each PEG molecule independently has a molecular weight of 5 KDa.

In some embodiments, the modified SDAB molecule comprises a linker of formula (I) linked to a PEG molecule and has a structure selected from:

In some embodiments, the modified SDAB molecule comprises a linker of formula (I) linked to a PEG molecule and has a structure selected from:
(Where each PEG molecule is independently a PEG monomer, polymer, or derivative thereof). In some embodiments, each PEG molecule is an mPEG monomer, polymer, or derivative thereof.

In some embodiments, the linker of formula (I) is linked to a PEG molecule represented by the following formula:

In some embodiments, the linker of formula (I) is linked to a PEG molecule represented by the following formula:

In some embodiments, the linker of formula (I) is linked to a PEG molecule represented by the following formula:

The linker-PEG molecule can associate with (eg, associate with) the SDAB molecule, thereby forming a modified SDAB molecule. Single domain molecules of the SDAB molecule may be arranged from N-terminus to C-terminus in the following order: TNFα-linked single domain molecule-TNFα-linked single domain molecule-PEG molecule (eg, branched PEG molecule ). In one embodiment, the modified SDAB molecule is represented by the following formula:

In one embodiment, the modified SDAB molecule is represented by the following formula:

In one embodiment, the modified SDAB molecule is represented by the following formula:

One exemplary embodiment of a modified SDAB molecule is represented by the following formula:

  The reactive group of the SDAB molecule is typically attached via a nucleophilic moiety attached to the SDAB molecule. In some embodiments, the nucleophilic moiety is sulfur (eg, the sulfur of a cysteine residue). In other embodiments, the nucleophilic moiety is nitrogen (eg, the nitrogen of the terminal α-amino group or the nitrogen-containing amino acid side chain (eg, the ε-amino group of the lysine chain)). In other embodiments, the nucleophilic moiety is a C-terminal group. The reactive group of the SDAB molecule is generally attached via an electrophilic moiety attached to the linker. In some embodiments, the electrophilic moiety is a carbonyl group (eg, an activated ester or aldehyde). In some embodiments, the electrophilic moiety is a maleimide group.

In another aspect, the invention features a method of making a modified SDAB molecule described herein. The method involves preparing an SDAB molecule (eg, obtaining the SDAB molecule from a cell culture (eg, a recombinant cell culture)) and attaching an SDAB molecule (eg, a single antigen binding domain) or a linker (eg, an SDAB molecule). The peptide linker) to the linker moiety of formula (I) wherein Y, X, W ¹ , W ² , Z, R ¹ , R ² , R ³ , m, n and p are as described above. Contacting under conditions under which at least one chemical bond is formed.

In some embodiments, Y is a bond. In some embodiments, Y is pyrrolidine-2,5-dione. In some embodiments, Y is C _1-4 alkylene substituted with 0-2 R ^a . In some embodiments, Y is C _1-4 alkylene substituted with 1 R ^a . In some embodiments, Y is methylene substituted with 1 R ^a . In some embodiments, R ^a is hydroxyl.

  In some embodiments, X is a bond. In some embodiments, X is oxygen (O). In some embodiments, X is not present.

In some embodiments, R ² is (a).

In some embodiments, R ² is (g).

In some embodiments, W ¹ is a bond. In some embodiments, W ¹ is NR ¹ .

In some embodiments, W ² is a bond. In some embodiments, W ² is NR ¹ .

In some embodiments, R ¹ is hydrogen.

  In some embodiments, Z is O, S or a bond.

  In some embodiments, Z is O.

In some embodiments, R ³ is hydrogen.

  In some embodiments, m is 0. In some embodiments, m is 1.

  In some embodiments, n is 0. In some embodiments, n is 2. In some embodiments, n is 3.

  In some embodiments, p is 0. In some embodiments, p is 3.

  In some embodiments, the SDAB molecule is linked via a cysteine residue.

  In some embodiments, treatment with the linker moiety of formula (I) is performed after reduction of the SDAB molecule. In some embodiments, the SDAB molecule is reduced to remove disulfide bridges formed between cysteine residues.

In some embodiments, the linker of formula (I) is linked to a PEG molecule represented by the following formula:

In one embodiment, the modified SDAB molecule is represented by the following formula:

  In another aspect, the invention features a composition, eg, a pharmaceutical composition, comprising a modified SDAB molecule described herein and a pharmaceutically acceptable carrier. The composition comprises a second agent, such as a TNFα-related disorder such as rheumatoid arthritis (RA) (eg moderate to severe rheumatoid arthritis), an arthritic condition (eg psoriatic arthritis, polyarticular juvenile idiopathic arthritis (JIA)). )), Treating ankylosing spondylitis (AS), psoriasis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, and / or multiple sclerosis, or treating inflammatory or autoimmune disorders A therapeutically or pharmacologically active second agent useful to do so can also be included.

  In yet another aspect, the invention provides a method of improving an inflammatory condition or autoimmune condition in a subject, eg, treating a TNFα-related disorder, such as an inflammatory disorder or an autoimmune disorder, in a subject (eg, a human subject). Or a method of prevention. Examples of TNFα-related disorders include rheumatoid arthritis (RA) (eg moderate to severe rheumatoid arthritis), arthritic conditions (eg psoriatic arthritis, polyarticular juvenile idiopathic arthritis (JIA)), ankylosing spondylitis ( AS), psoriasis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, and / or multiple sclerosis. The method comprises subjecting a subject, eg, a human patient, with a TNFα-binding modified SDAB molecule described herein alone or TNFα-related in an amount such that one or more of the symptoms of a TNFα-related disorder is alleviated. Administration in conjunction with a therapeutically or pharmacologically active second agent useful for treating the disorder.

  In one embodiment, a modified SDAB molecule (eg, a composition containing a modified SDAB molecule) described herein is provided to a subject, eg, a human subject (eg, a patient having a TNFα-related disorder). Suitable for administration. SDAB molecules can be administered to a subject by injection (eg, subcutaneous injection, vascular injection, intramuscular injection or intraperitoneal injection) or by inhalation.

  In certain embodiments, the modified SDAB molecule and the second agent are administered together, eg, simultaneously or sequentially. In one embodiment, the modified SDAB molecule and the second agent are administered in the same composition, eg, a pharmaceutical composition described herein. In one embodiment, the second agent is an anti-TNFα antibody molecule or a TNFα binding fragment thereof, and the second TNFα antibody is an epitope that differs from the TNFα-binding modified SDAB molecule described herein. Combine with. Other non-limiting examples of second agents that can be co-administered or co-formulated with a TNFα-binding modified SDAB molecule include cytokine inhibitors, growth factor inhibitors, immunosuppressants, anti- Examples include, but are not limited to, inflammatory agents, metabolic inhibitors, enzyme inhibitors, cytotoxic agents, and cytostatic agents. In one embodiment, the additional agent is a standard treatment for arthritis, including non-steroidal anti-inflammatory drugs (NSAIDs); corticosteroids (including prednisolone, prednisone, cortisone and triamcinolone) And disease-modifying anti-rheumatic drugs (DMARDs), such as methotrexate, hydroxychloroquine (Plaquenil) and sulfasalazine, leflunomide (Arava ™), tumor necrosis factor inhibitors (Etanercept (Embrel ™), infliximab ™ (Remicade ™); )) (With or without methotrexate) and adalimumab (Humira ™)), anti-CD20 antibodies (eg Rituxan ™), soluble interleukin-1 receptors such as anakinra ( Ineret (TM)), gold, minocycline (Minocin (R)), penicillamine, and cytotoxic agents (azathioprine, including cyclophosphamide and cyclosporine) include, but are not limited to. Beneficially, in such combination therapy, the therapeutic agent to be administered can be used at lower doses, thus avoiding the toxicities or complications that can occur in connection with various monotherapy. Alternative combinations of excipients and / or second therapeutic agents can be identified and tested according to the instructions provided herein.

  In another aspect, the invention features a method of evaluating a modified SDAB molecule, eg, a modified SDAB molecule described herein. The method comprises administering a modified SDAB molecule described herein to a subject, eg, a human subject (eg, a patient having a TNFα-related disorder) and one or more drugs of the modified SDAB molecule. Evaluating kinetic / pharmacodynamic (PK / PD) parameters. SDAB molecules can be administered to a subject by injection (eg, subcutaneous injection, vascular injection, intramuscular injection or intraperitoneal injection) or by inhalation.

In a related aspect, the invention features a method of evaluating or selecting a modified SDAB molecule (eg, a modified TNFα-binding SDAB molecule described herein). This method
Presenting a test value, eg, an average test value, for at least one PK / PD parameter of the SDAB molecule in a subject, eg, a human or animal subject;
Comparing the presented test value, eg, the average test value, with at least one reference value, thereby evaluating or selecting an SDAB molecule;
including.

  In some embodiments, presenting the test value comprises obtaining a sample of a SDAB molecule, eg, a sample batch of an antibody cell culture, and / or after modification of the SDAB molecule, the pharmacokinetics described herein. Testing for at least one of the parameters. The methods disclosed herein can be useful from a process perspective, for example, to monitor or ensure consistency or quality between batches.

  In certain embodiments, a method for evaluating a modified SDAB molecule comprises preparing a sample, eg, a sample containing a modified SDAB molecule, and a capture detection assay, eg, a protein detection assay or herein. And testing the sample with a whole molecule detection assay as described in Example 11b. In one embodiment, the sample is contacted with a target immobilized on a solid support (eg, a biotinylated target molecule associated with conjugated streptavidin) and the bound SDAB-target molecule complex is modified with a modified SDAB. Detection is performed using a reagent that binds to the protein portion of the molecule, such as an antibody. In such an assay format, the protein portion of the modified SDAB molecule is detected. In other embodiments, the sample is contacted with a target immobilized on a solid support (eg, a biotinylated target molecule associated with conjugated streptavidin) and the bound SDAB-target molecule complex is modified with a modified SDAB. Detection is performed using a reagent, such as an antibody, that binds to the polymer (eg, PEG) portion of the molecule. In such embodiments, the polymer (eg, PEGylated) portion of the SDAB molecule is detected. Preferably, detection of the polymer (eg, PEG) moiety captures the entire modified SDAB-polymer conjugate because no unconjugated SDAB molecules are detected.

The PK / PD parameters evaluated by this method are: in vivo concentrations of modified SDAB molecules (eg, concentrations in blood, serum, plasma and / or tissue); clearance (CL) of modified SDAB molecules; SDAB molecule volume distribution (V _dss or Vc); modified SDAB molecule half-life (t _1/2 ); modified SDAB molecule bioavailability; modified SDAB molecule maximum blood concentration, maximum serum Concentration, maximum plasma concentration, or maximum tissue concentration; modified SDAB molecule exposure (AUC = area under the concentration time curve); modified SDAB molecule tissue to serum, tissue to plasma, or tissue to blood AUC ratio or concentration ratio of: a modified SDAB molecule intact or degradation product urine concentration; or serum, Whey or tissue in free form can be selected linked and from one or more of the total target concentration.

  In one embodiment, one or more PK / PD parameters are evaluated at one, two, or more predetermined time intervals after administering the modified SDAB molecule to the subject. In one embodiment, at least one PK / PD parameter of a modified SDAB molecule is altered, eg, improved, compared to a reference standard, eg, an unmodified SDAB molecule. For example, a modified SDAB molecule has an increased half-life and / or bioavailability compared to an unmodified SDAB molecule; has a different tissue distribution (eg, localization to a different tissue or organ (eg, small intestine or large intestine)) Have one or more. In certain embodiments, the PK / PD parameters are used to provide an effective value or suitability criteria for treatment. Other measures of efficacy, including but not limited to, improving one or more symptoms, improving quality of life, reducing inflammatory markers, can also be made as part of the efficacy assessment.

  In some embodiments, one or more PK / PD parameters, valid values, or an indication of whether a pre-selected validity criterion is met is recorded or stored, for example, on a computer readable medium . Indicators that meet such values or pre-selected efficacy criteria are, for example, product descriptions, official documents (eg, US Pharmacopoeia), for commercial use or for submission to US or foreign regulatory authorities, or It can be written on any other material that can be distributed, such as a label.

  In another aspect, the invention features a detection method or capture detection assay, such as a protein detection assay or a whole molecule detection assay as described in Example 11b herein. The method or assay involves preparing a sample containing a modified SDAB molecule (eg, obtaining a sample obtained from a subject after administration of the SDAB molecule) and immobilizing the sample on a solid support. Contacting (eg, TNFα) (eg, a biotinylated target molecule associated with conjugated streptavidin) and binding the bound SDAB-target complex to the protein or polymer (eg, PEG) portion of the modified SDAB molecule Detecting using a reagent such as an antibody. In an assay format where the reagent binds to the protein portion of the SDAB molecule, the protein portion of the modified SDAB molecule is detected. In an assay format where the reagent binds to the modified PEG moiety of the SDAB molecule, the polymer (eg, PEGylated) moiety of the SDAB molecule is detected. Preferably, detection of the PEG moiety captures the entire modified SDAB-polymer conjugate because no SDAB molecules that are not conjugated are detected.

  In another aspect, the invention features a kit or article of manufacture that includes a device, syringe, or vial containing an SDAB molecule or a composition described herein. The kit or product may optionally include instructions for use. In certain embodiments, the syringe or vial is constructed of glass, plastic or a polymer material, such as a cyclic olefin polymer or copolymer. In other embodiments, the formulation can be present in an injection device (eg, an injection syringe, eg, a pre-filled injection syringe). The syringe can be adapted for administration by an individual, for example, as a single vial system containing a self-injector (eg, a pen injector device) and / or instructions for use. In one embodiment, the injection device is a filled pen self-injection device or other suitable self-injection device, optionally with instructions for use and administration.

  In certain embodiments, a kit or product pre-packaged with instructions for administration (eg, self-administration) by injection (eg, subcutaneous injection, vascular injection, intramuscular injection, intra-articular injection or intraperitoneal injection) (E.g., single or multiple dose filled pre-filled syringes or syringes) are provided to a subject, such as a patient or health care provider.

  In other embodiments, the invention features devices for nasal, transdermal, and intravenous administration of the formulations described herein. For example, transdermal patches are provided for administration of the formulations described herein. In still other cases, an intravenous bag is provided for administration of the formulations described herein. In some embodiments, an intravenous bag containing saline or 5% dextrose is provided.

  In another aspect, the invention provides a method for administering a modified SDAB molecule or composition described herein to a patient (eg, a human patient) in need of a modified SDAB molecule, eg, a TNFα SDAB molecule. It is characterized by a method of indicating whether to do. The method comprises (i) providing the patient with at least one unit dose of an SDAB molecule as described herein; and (ii) providing the patient with an injection (eg, subcutaneous injection, vascular injection, intramuscular injection or intraperitoneal injection). Directing at least one unit dose to self-administer. In one embodiment, the patient has a TNFα-related disorder, such as an inflammatory disorder or an autoimmune disorder as described herein.

  In another aspect, the invention features a method of instructing a recipient to administer a modified SDAB molecule described herein. The method describes how to administer the formulation to a patient (eg, end user, patient, physician, practitioner or wholesaler, distributor, or hospital in-house pharmacy, nursing home clinic, or HMO). Including instructing what to do.

  In another aspect, a method of distributing a modified SDAB molecule described herein is provided. The method can be used to treat a patient for at least 6 months, 12 months, 24 months, or 36 months, such as a recipient (e.g., end user, patient, physician, practitioner or wholesaler, distributor, or hospital pharmacy, Providing a package containing a sufficient unit dose of SDAB molecules to a nursing home clinic or HMO).

  In another aspect, the present invention provides the quality of a package or package lot of a formulation described herein containing a modified SDAB molecule described herein (eg, whether the package has expired). Features a method or process to be evaluated (to determine if). The method includes evaluating whether the package has expired. The expiration date is a preselected event, such as at least 6 months, 12 months, 24 months, 36 months or 48 months from manufacture, inspection or packaging, for example, more than 24 months or 36 months. In some embodiments, the results of the analysis are used to make a decision or take measures, such as using or discarding SDAB molecules in a package depending on whether the product has expired or not. To be classified, selected, released or refrained from shipping, shipped, moved to a new location, put on the market, sold or planned for sale, collected from the market, or Cancel the sales plan.

  In another aspect, the invention features a method that complies with regulatory requirements, eg, post-approval requirements of regulatory authorities, eg, FDA. The method includes providing an evaluation of the antibody formulation with respect to the parameters as described herein. Post-approval requirements may include one or more criteria for the above parameters. The method determines whether an optionally observed solution parameter meets a preselected criterion, or whether the parameter is within a preselected range; optionally the value or result of the analysis Or optionally communicating with the regulatory authority, for example by transmitting its value or result to the regulatory authority.

  In another aspect, the invention provides a modified SDAB molecule, eg, TNFα SDAB, having a preselected property, eg, a property described herein, eg, satisfying a sales specification, labeling requirement, or official requirement. Features a method of making a batch of molecules. The method comprises preparing a test sample containing a modified SDAB molecule, analyzing the test sample according to the methods described herein, and wherein the test formulation meets preselected criteria. For example, determining whether to have a preselected relationship with a reference value, eg, one or more reference values disclosed herein, and preparing test samples to create a batch of products Selecting an object.

  In another aspect, the invention provides a composite batch of a blend of modified SDAB molecules, eg, TNFα SDAB molecules, wherein one or more parameters for each batch (eg, as determined by the methods described herein). Combined batches of formulations in which a desired value or solution parameter) changes from a preselected desired reference value or standard, eg, a range or standard described herein, to a less than preselected range It is characterized by. In some embodiments, one or more parameters for one or more batches of the formulation are determined, and the batch (s) are selected in response to the determination. Some embodiments include comparing the result of the determination to a preselected value or criterion, eg, a reference criterion. Other embodiments include adjusting the dose of the batch to be administered, for example based on the results of the determination of values or parameters.

  Patent publications, patent applications, patents and other references mentioned herein are hereby incorporated by reference in their entirety.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

  Other features and advantages of the invention will be apparent from the detailed description, drawings, and claims.

It is a figure which shows the amino acid sequence (sequence number 1) of SDAB-01. Bold CDRs correspond to single antigen binding domain components, each having the amino acid sequence of amino acids 1-115 of SEQ ID NO: 1. The flexible linker is shown in lower case. The modified C-terminal cysteine that supports site-specific PEGylation is also shown in bold. It is a figure which shows the polyethyleneglycol (PEG) (molecular weight 40000, 2 * 20 kDa) used for SDAB-01 molecule | numerator. The PEG activating group is maleimide. It is the schematic of SDAB-01. A shows the structures of linear mPEG-maleimide (Control 2) and two branched mPEG-maleimides ([SEQ ID NO: 1] -PEG40 and SDAB-01). B is a scan diagram comparing the sizes of SDAB-01 and [SEQ ID NO: 1] -PEG40. FIG. 2 is a FACS (“fluorescence activated cell classification”) scan diagram of cell surface staining of SDAB-01 on CHO-TNF-D13 (pW2128) cells expressing membrane bound TNFα. Cells were stained sequentially with SDAB-01, biotinylated anti-PEG and streptavidin-PE (grey area) or pseudostained with streptavidin-PE (white area). FIG. 2 shows a dose response curve of SDAB-01 compared to control 3 and control 4 of non-PEGylated SDAB polypeptide in a cytotoxicity assay using human TNFα or rhesus monkey TNFα. It is a figure which shows the TNF alpha binding curve with respect to SDAB-01. Various concentrations of (a) human, (b) rhesus macaque, (c) rat and (d) mouse TNFα ranging from 0.195 nM to 100 nM, and (e) 0.195 nM to 400 nM. Rabbit TNFα was injected against SDAB-01 immobilized. Each data set represents at least two independent experiments. 2 is a graph showing the effect of SDAB-01 on total leukocyte infiltration in a murine air sac model in Experiment 1. FIG. 2 is a graph showing the effect of SDAB-01 on neutrophil infiltration in a murine air sac model in Experiment 1. FIG. It is a graph which shows the effect of SDAB-01 with respect to the total leukocyte infiltration in the murine air sac model in Experiment 2. It is a graph which shows the effect of SDAB-01 with respect to the neutrophil infiltration in the murine air sac model in Experiment 2. It is a graph which shows the effect of SDAB-01 with respect to the total leukocyte infiltration in the murine air sac model in Experiment 3. It is a graph which shows the effect of SDAB-01 with respect to neutrophil infiltration in the murine air sac model in Experiment 3. 10 mg / kg, 3 mg / kg, 1 mg / kg, 0.3 mg / kg, 0.1 mg / kg SDAB-01, 1 mg / kg control SDAB, 10 mg / kg and 3 mg / kg infliximab, 10 mg / kg control FIG. 2 is a graph showing weekly body weights for animals that received treatment with antibody or 10 ml / kg vehicle twice a week. 10 mg / kg, 3 mg / kg, 1 mg / kg, 0.3 mg / kg, 0.1 mg / kg SDAB-01, 1 mg / kg control SDAB, 10 mg / kg and 3 mg / kg infliximab, 10 mg / kg control 2 is a graph showing the weekly mean disease severity score in animals that received treatment with antibody or vehicle at 10 ml / kg twice a week. 10 mg / kg, 3 mg / kg, 1 mg / kg, 0.3 mg / kg, 0.1 mg / kg SDAB-01, 1 mg / kg control SDAB, 10 mg / kg and 3 mg / kg infliximab, 10 mg / kg control It is a graph which shows the disease severity of the 7th week after the process by the animal which administered the antibody or the vehicle of 10 ml / kg twice a week. 10 mg / kg, 3 mg / kg, 1 mg / kg, 0.3 mg / kg, 0.1 mg / kg SDAB-01, 1 mg / kg control SDAB, 10 mg / kg and 3 mg / kg infliximab, 10 mg / kg control It is a graph which shows the group average severity score using the microscope of the 7th week after the process by the animal which administered the antibody or the vehicle of 10 ml / kg twice a week. 10 mg / kg, 3 mg / kg, 1 mg / kg, 0.3 mg / kg, 0.1 mg / kg SDAB-01, 1 mg / kg control SDAB, 10 mg / kg and 3 mg / kg infliximab, 10 mg / kg control It is a graph which shows the comparison with the group average severity score and disease severity score using the microscope of the 7th week after the process in the animal which administered the antibody or the vehicle of 10 ml / kg twice a week. 10 mg / kg, 3 mg / kg, 1 mg / kg, 0.3 mg / kg, 0.1 mg / kg, 0.03 mg / kg SDAB-01, 1 mg / kg control SDAB, 10 mg / kg and 3 mg / kg infliximab FIG. 5 is a graph showing weekly body weights in animals that received treatment with 10 mg / kg control antibody or 10 ml / kg vehicle twice a week. 10 mg / kg, 3 mg / kg, 1 mg / kg, 0.3 mg / kg, 0.1 mg / kg, 0.03 mg / kg SDAB-01, 1 mg / kg control SDAB, 10 mg / kg and 3 mg / kg infliximab 2 is a graph showing the weekly mean disease severity score in animals that received twice a week treatment with 10 mg / kg control antibody or 10 ml / kg vehicle. 10 mg / kg, 3 mg / kg, 1 mg / kg, 0.3 mg / kg, 0.1 mg / kg, 0.03 mg / kg SDAB-01, 1 mg / kg control SDAB, 10 mg / kg and 3 mg / kg infliximab FIG. 7 is a graph showing disease severity scores at 7 weeks after treatment in animals administered 10 mg / kg control antibody or 10 ml / kg vehicle twice a week. FIG. 10 mg / kg, 3 mg / kg, 1 mg / kg, 0.3 mg / kg, 0.1 mg / kg, 0.03 mg / kg SDAB-01, 1 mg / kg control SDAB, 10 mg / kg and 3 mg / kg infliximab FIG. 5 is a graph showing group mean severity scores using a microscope after treatment twice a week with 10 mg / kg control antibody or 10 ml / kg vehicle. 10 mg / kg, 3 mg / kg, 1 mg / kg, 0.3 mg / kg, 0.1 mg / kg, 0.03 mg / kg SDAB-01, 1 mg / kg control SDAB, 10 mg / kg and 3 mg / kg infliximab Shows comparison of group mean severity score and disease severity score using microscope at 7 weeks after treatment in animals administered 10 mg / kg control antibody or 10 ml / kg vehicle twice a week It is a graph. FIG. 6 is a graph showing the mean (± SD) of SDAB-01 serum concentration-time profiles after 3 mg / kg single IV administration or single SC administration in male cynomolgus monkeys. FIG. 6 is a graph showing the mean (± SD) of serum concentrations normalized by the dose of PEGylated TNFα SDAB polypeptide after a single IV administration to mice, rats or cynomolgus monkeys. TNFα SDAB polypeptide 2 × 20 kDa PEG (black circle), TNFα SDAB polypeptide 4 × 10 kDa PEG (white circle), or TNFα SDAB polypeptide 1 × 40 kDa PEG (black triangle) in a single IV bolus administration to B6CBAF1 / J mice (A 2 mg / kg for the 2 × 20 kDa PEG conjugate and 3 mg / kg for the other two conjugates), Sprague Dawley rats (B; 2 mg / kg), or cynomolgus monkeys (C; 3 mg / kg). In mouse and monkey PK studies (A and C), unlabeled test substances were used, and in rat PK studies (B), 125I-labeled test substances were used. Single sampling was used for mice (n = 3 per time point) and sequential sampling was used for rats (n = 5-7 per compound) and monkeys (n = 3 per compound). Serum concentration was determined by specific immunoassay (mouse and monkey, in ng / mL) or gamma ray measurement (rat, ng x equivalent / mL). The lifetime was 14 days, 24 days and 56 days to 62 days for mice, rats and monkeys, respectively. Individual animal concentration values below the limit of quantification (LOQ) were treated as zero for the calculation of mean and SD. Data show the mean (± SD) of concentrations normalized at each time point (ie at a 1 mg / kg dose). Data points for mean serum concentration of 0 ng / mL (ie below LOQ in all animals) are not shown on a log scale. FIG. 2 is a graph showing the mean tissue exposure and mean serum exposure (AUC0-168 hr) of 125I-labeled PEGylated TNFα SDAB polypeptide after a single IV dose of 0.3 mg / kg to mice. B6CBAF1 / J mice received 125I-labeled TNFα SDAB molecule branched 2 × 20 kDa PEG (black bar) or TNFα SDAB molecule linear 40 kDa PEG (gray bar) at a single IV bolus dose of 0.3 mg / kg. Administered. Serum and tissue samples (n = 8-12 per time point) were collected over 7 days (168 hours) and radioequivalent (RE) concentrations in the tissue and serum were determined by gamma counting. Serum (μg × equivalent / mL unit) and AUC 0-168 hr (area under the time curve of time 0-168 hours) in each tissue (μg × equivalent / g unit) were measured using the sparse sampling method. Determined by compartmental analysis, a 95% confidence interval (95% CI, error bars on the graph) was calculated using the standard error of the mean. An asterisk (*) indicates a statistically significant difference of AUC 0 to 168 hr between the two constructs (p <0.05). 1 is a graph showing a cation exchange high performance liquid chromatography (CEX-HPLC) profile of PEGylated TNFα SDAB polypeptide. The protein concentration of each material was adjusted to 1.0 mg / mL using formulation buffer and 10 μL was injected onto a Dionex ProPac WCX-10 column. Mobile phase A was 10 mM ammonium formate (pH 4.0). Mobile phase B was 10 mM ammonium formate, 500 mM sodium chloride (pH 4.0). The protein conjugate was eluted with a linear gradient of sodium chloride (B from 0% to 40% in 40 minutes) at a flow rate of 0.75 mL / min. Absorbance at 280 nm was monitored. 1 is a graph showing a size exclusion high performance liquid chromatography (SEC-MALS) profile by multi-angle light scattering of a PEGylated TNFα SDAB polypeptide. Dilute TNFα SDAB polypeptide 2 × 20 kDa PEG (dashed line), TNFα SDAB polypeptide 4 × 10 kDa PEG (dotted line), or TNFα SDAB polypeptide 1 × 40 kDa PEG (solid line) to 2.0 mg / mL, 100 μL was injected onto a Superose 6 mobile phase column maintained at 30 ° C. Retention time (diagram), total mass (black circles), PEG mass (white triangles) and protein mass (x) were determined using Wyatt Technologies' ASTRA V v5.4.14. FIG. 5 is a graph showing the determination of hydrodynamic radius (Rh) and root mean square radius (RMS or Rg). FIG. 2.0 mg of TNFα SDAB polypeptide 2 × 20 kDa PEG (dashed line and white square), TNFα SDAB polypeptide 4 × 10 kDa PEG (green line and symbol), or TNFα SDAB polypeptide 1 × 40 kDa PEG (dotted line and white triangle) Each sample was injected onto a Superose 6 mobile phase column maintained at 100 μL and 30 ° C. Analysis of retention time (solid line and black circles), Rh (A) and RMS (B) was performed using ASTRA V v5.4.14 from Wyatt Technologies. ADCC activity of Control 1, Control 2, Control 3, and Control IgG1 antibodies compared to ADCB activity of SDAB-01 using CHO-TNFD13 (pW2128) cells labeled with CSFE as target and human NK cells as effector It is a graph which shows. The value of ADCC activity (%) is calculated as the percentage of target cells that are 7AAD +. The plotted values are 7 AAD + target cells (%) using the test drug minus 7 AAD + target cells (%) in the presence of effector cells alone. This plot is representative of the four individual ADCC assays that were performed and demonstrated that SDAB-01 had no ADCC activity. Shows CDC activity of Control 1, Control 2, Control 3, and Control IgG1 antibodies compared to CDC activity of SDAB-01 against CHO-TNF-D13 (pW2128) strain in the presence of rabbit complement in vitro It is a graph. Cytotoxicity was assessed by 7AAD uptake by dead cells and the plotted values are the% of 7AAD + cells in the presence of complement alone minus the% of 7AAD + cells with test drug and control. Samples were made in duplicate for adalimumab, infliximab and SDAB-01. This plot is representative of the three individual assays performed and demonstrated that SDAB-01 has no CDC activity.

  The present invention relates to modified single domain antigen binding molecules (also referred to herein as “SDAB molecules”). A modified SDAB molecule can include one or more single antigen binding domains that interact with one or more targets, eg, bind to one or more targets. In one embodiment, one or more of the single antigen binding domains of the modified SDAB molecule binds to tumor necrosis factor-α (TNFα). An SDAB molecule can be modified to increase its biological properties in vivo. For example, an SDAB molecule has one or more of increased half-life, reduced immunogenicity, or improved at least one pharmacokinetic / pharmacodynamic (PK / PD) parameter compared to an unmodified SDAB molecule. It can be modified to improve. In one embodiment, the modified SDAB molecule includes one or more polymer molecules, such as poly (ethylene glycol) (PEG) or derivatives thereof. Modified SDAB molecules are useful, for example, for administration to a subject, eg, a human. Also disclosed are methods of preparing and using modified SDAB molecules for treating or preventing TNFα-related disorders.

  In order to more easily understand the present invention, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

  As used herein, the articles “a” and “an” refer to one or more (eg, at least one) grammatical objects of an article.

  The term “or” is used herein to mean the term “and / or” and is used interchangeably herein unless the context clearly indicates otherwise.

  The terms “protein” and “polypeptide” are used interchangeably herein.

  “About” and “approximately” shall generally mean an acceptable degree of error to the quantity being measured given the nature or accuracy of the measurement. Examples of the degree of error are within 20 percent (%) of a given value or range of values, typically within 10%, more typically within 5%.

  In the context of a nucleotide sequence, the term “substantially identical” refers to a polypeptide in which a first nucleotide sequence and a second nucleotide sequence have a common functional activity or a common structural polymorphism. To a first nucleic acid sequence, eg, a reference sequence, that contains a sufficient or minimal number of nucleotides identical to nucleotides aligned to a second nucleic acid sequence to encode a peptide domain or common functional polypeptide activity To represent a nucleotide sequence having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Used.

  The polypeptides of the present invention also include fragments, derivatives, analogs or variants of the above polypeptides, and any combinations thereof. When referring to a protein of the invention, the terms “fragment”, “variant”, “derivative” and “analog” are any that retain at least some of the functional properties of the corresponding natural antibody or polypeptide. Including polypeptides. Fragments of the polypeptides of the present invention include specific antibody fragments described elsewhere herein in addition to proteolytic and deletion fragments. The variants of the polypeptide of the present invention include, in addition to the above fragments, polypeptides in which the amino acid sequence has been changed by amino acid substitution, deletion or insertion. Variants may be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be generated using mutagenesis techniques known in the art. Variant polypeptides can include conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives of the fragments of the present invention are polypeptides that have been altered to exhibit additional characteristics not found in natural polypeptides. An example is a fusion protein. Variant polypeptides can also be referred to herein as “polypeptide analogs”. As used herein, a “derivative” of a polypeptide refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. “Derivatives” also includes those polypeptides that contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, proline can be replaced with 4-hydroxyproline, lysine can be replaced with 5-hydroxylysine, histidine can be replaced with 3-methylhistidine, and serine can be replaced with homoserine. The lysine can be replaced with ornithine.

  The term “functional variant” has an amino acid sequence that is substantially identical to a naturally occurring sequence or is encoded by a substantially identical nucleotide sequence, and one or more activities of the naturally occurring sequence. Refers to a polypeptide that can have

  Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.

  To determine the identity (percentage) of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., the first amino acid sequence or nucleic acid sequence and the first of the gaps for optimal alignment). Two amino acid sequences or one or both of the nucleic acid sequences can be introduced, and heterologous sequences can be ignored for comparison purposes). In exemplary embodiments, the length of the reference sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50% or 60%, or at least 70%, 80%, 90% of the length of the reference sequence. % Or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, an amino acid or Nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).

  The percent identity between two sequences is the same shared by those sequences, taking into account the number of gaps and the length of each gap, which must be introduced for optimal alignment of the two sequences. It depends on the number of positions.

  Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the percent identity between two amino acid sequences is either a Blossum 62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6 or 4, and 1, 2, Needleman and Wunsch ((1970) J. Mol. Biol. 48) incorporated into the GAP program in the GCG software package (available at www.gcg.com) with 3, 4, 5 or 6 length weights. : 444-453) determined using an algorithm. In yet another embodiment, the percent identity between two nucleotide sequences is determined by NWSgapdna. GCG software package (available at www.gcg.com) using CMP matrix, 40, 50, 60, 70 or 80 gap weights and 1, 2, 3, 4, 5 or 6 length weights In the GAP program. One exemplary set of parameters (used unless otherwise specified) is Blossum 62 with a gap gap penalty of 12, a gap extend penalty of 4, and a frame shift gap penalty of 5. It is.

  The percent identity between two amino acid or nucleotide sequences is incorporated into the ALIGN program (version 2.0) using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. It can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4: 11-17).

  The nucleic acid and protein sequences described herein can be used as “query sequences” to perform public database searches, eg, to identify other family members or related sequences. Such a search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215: 403-10. A BLAST nucleotide search can be performed with the NBLAST program, score = 100, word length = 12, to obtain a nucleotide sequence homologous to the nucleic acid molecule characterized in the present invention. A BLAST protein search is performed with the XBLAST program, score = 50, word length = 3, and an amino acid sequence homologous to the protein (SEQ ID NO: 1) molecule characterized in the present invention can be obtained. To obtain a gapped alignment for comparative purposes, gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25: 3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of each program (eg, XBLAST and NBLAST) can be used.

  A “conservative amino acid substitution” is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. A family of amino acid residues having similar side chains has been defined in the art. These families include amino acids with basic side chains (eg lysine, arginine, histidine), amino acids with acidic side chains (eg aspartic acid, glutamic acid), amino acids with uncharged polar side chains (eg glycine, asparagine, Glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (eg threonine, valine, Isoleucine) and amino acids having aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine).

  Various aspects of the invention are described in further detail below.

Single Domain Antigen Binding (SDAB) Molecules Single domain antigen binding (SDAB) molecules include molecules in which the complementarity determining region is part of a single domain polypeptide. Examples include heavy chain variable domains, binding molecules that naturally lack a light chain, Nanobody ™, single domains derived from conventional four-chain antibodies, modified domains, and those other than those derived from antibodies Single domain scaffolds include but are not limited to them. The SDAB molecule can be any in the art or any future single domain molecule. SDAB molecules can be derived from any species including, but not limited to, mouse, human, camel, llama, fish, shark, goat, rabbit and cow. The term also includes naturally occurring single domain antibody molecules from species other than camelids and sharks.

  In one aspect, the SDAB molecule may be derived from an immunoglobulin variable region found in fish, such as derived from an immunoglobulin isotype known as a novel antigen receptor (NAR) found in shark serum, for example. it can. Methods for producing single domain molecules (“IgNAR”) derived from the variable region of NAR are described in WO 03/014161 and Streltsov (2005) Protein Sci. 14: 2901-2909.

  According to another aspect, the SDAB molecule is a naturally occurring single domain antigen binding molecule known as a heavy chain lacking a light chain. Such single domain molecules are disclosed, for example, in WO 9404678 and Hamers-Casterman, C. et al. (1993) Nature 363: 446-448. For clarity, this variable domain derived from a heavy chain molecule lacking the light chain in its natural state, known herein as VHH or Nanobody ™, is the conventional VH of the four-chain immunoglobulin. Distinguish. Such VHH molecules can be derived from camelid species such as camelid, llama, dromedary, alkapa and guanaco camelid species. Other species other than camelids can produce heavy chain molecules that lack the light chain in nature and such VHHs are within the scope of the present invention.

  SDAB molecules were generated recombinantly, CDR-grafted, humanized, camelized, deimmunized, and / or generated in vitro (eg, selected by phage display), as described in more detail below. Can be a thing.

  The term “antigen binding” is intended to include a portion of a polypeptide that includes determinants that form an interface that binds to a target antigen or epitope thereof, eg, a single domain molecule as described herein. In the context of a protein (or protein mimetic), an antigen binding site typically has one or more loops (at least four amino acids or one or more of amino acid mimetics) that form an interface that binds to the target antigen. Loop). Typically, an antigen binding site of a polypeptide, such as a single domain antibody molecule, comprises at least one or two CDRs, or more typically at least three, four, five or six CDRs.

  The term “immunoglobulin variable domain” is often understood in the art as being identical or substantially identical to a VL or VH domain of human or animal origin. It is recognized that immunoglobulin variable domains can be developed in certain species, such as sharks and llamas, to differ in amino acid sequence from human or mammalian VL or VH. However, these domains are mainly involved in antigen binding. The term “immunoglobulin variable domain” typically comprises at least one or two CDRs, or more typically at least three CDRs.

  A “constant immunoglobulin domain” or “constant region” is intended to include an immunoglobulin domain that is the same or substantially similar to CL, CH1, CH2, CH3 or CH4 which are domains of human or animal origin. See, for example, Charles A Hasemann and J. Donald Capra, Immunoglobulins: Structure and Function, in William E. Paul, ed., Fundamental Immunology, Second Edition, 209, 210-218 (1989). The term “Fc region” refers to a constant immunoglobulin domain comprising immunoglobulin domains CH2 and CH3, or an Fc portion of an immunoglobulin domain substantially similar thereto.

  In certain embodiments, the SDAB molecule is a monovalent or multispecific molecule (eg, a bivalent molecule, a trivalent molecule, or a tetravalent molecule). In other embodiments, the SDAB molecule is a monospecific molecule, a bispecific molecule, a trispecific molecule or a tetraspecific molecule. Whether a molecule is “monospecific” or “multispecific”, eg, “bispecific”, represents the number of different epitopes to which a binding polypeptide reacts. Multispecific molecules can be specific for different epitopes of the target polypeptides described herein, or specific for heterologous epitopes such as heterologous polypeptides or solid support materials in addition to the target polypeptide. It can be.

  As used herein, the term “valency” refers to the number of binding domains, eg, antigen binding domains, that may be present in an SDAB molecule. Each binding domain specifically binds to one epitope. When an SDAB molecule contains two or more binding domains, and each binding domain specifically binds to the same epitope, it is referred to as “bivalent monospecificity” for an antibody having two binding domains, or When each binding domain specifically binds to a different epitope, it is referred to as “bivalent bispecificity” for SDAB molecules having two binding domains. SDAB molecules are bispecific and can be bivalent for each specificity (referred to as “bispecific tetravalent molecules”). Bispecific bivalent molecules and methods for making them are described, for example, in US Pat. Nos. 5,731,168, 5,807,706, 5,821,333, and US Patent Application Publication No. 2003/020734 and 2002/0155537, the disclosures of all of which are incorporated herein by reference. Bispecific tetravalent molecules and methods for making them are described, for example, in WO 02/096948 and WO 00/44788, the disclosures of which are hereby incorporated by reference. Have been described. Generally, PCT International Publication No. 93/17715, International Publication No. 92/08802, International Publication No. 91/00360, International Publication No. 92/05793, Tutt et al., J. Immunol. 147: 60 -69 (1991), U.S. Pat.Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, 5,601,819 No., Kostelny et al., J. Immunol. 148: 1547-1553 (1992).

  In certain embodiments, the SDAB molecule comprises a complementary variable domain or one or more single domain molecules that lack an immunoglobulin constant (eg, Fc) region that binds to one or more target antigens. A single chain fusion polypeptide. An example of a target antigen recognized by an antigen-binding polypeptide is tumor necrosis factor α (TNFα). In certain embodiments, an antigen-binding single domain molecule is modified by associating, eg, covalently attaching, the domain with a PEG, eg, a branched PEG molecule.

TNFα
Tumor necrosis factor alpha is known in the art to be associated with inflammatory disorders such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis. Both TNFα and the receptors (CD120a and CD120b) have been studied in great detail. TNFα is a trimer in its biologically active form. Several strategies have been developed to antagonize the action of TNFα using anti-TNFα antibodies, and Remicade ™ and Humira ™ are now commercially available. Antibody molecules against TNFα are known. Many examples of TNFα-binding single domain antigen binding molecules are described in WO 2004/041862, WO 2004/041865, WO 2006/122786 (the contents of which are incorporated herein by reference in their entirety). Which is part of the specification). Further examples of single domain antigen binding molecules include U.S. Patent Application Publication No. 2006/0286066, U.S. Patent Application Publication No. 2008/0260757, International Publication No. WO 06/003388, U.S. Patent Application Publication No. 05/0271663, No. 06/0106203, the entire contents of which are hereby incorporated by reference. In other embodiments, monospecific, bispecific, trispecific and other multispecific single domain antibodies and PEG to TNFα.

  As used herein, the terms “TNF”, “TNFα” and “TNF-alpha” are used interchangeably and have the same meaning.

  In certain embodiments, the TNFα-binding SDAB molecule comprises one or more of the SDAB molecules disclosed in Table 11 herein and WO 2006/122786. For example, the TNFα-binding SDAB molecule can be a monovalent, divalent, or trivalent TNFα-binding SDAB molecule disclosed in WO 2006/122786. Examples of TNFα-binding SDAB molecules include, but are not limited to, TNF1, TNF2, TNF3, and their humanized forms (eg, TNF29, TNF30, TNF31, TNF32, TNF33). Further examples of monovalent TNFα-binding SDAB molecules are disclosed in Table 8 of WO 2006/122786. Examples of bivalent TNFα-binding SDAB molecules include two TNF30 SDAB molecules linked via a peptide linker to form a single fusion polypeptide, including TNF55 and TNF56 (WO 2006). Disclosed in US Pat. No. 1,222,786), but is not limited thereto. Further examples of bivalent TNFα-binding SDAB molecules are disclosed in Table 19 of WO2006 / 122786 as TNF4, TNF5, TNF6, TNF7, TNF8).

In other embodiments, two or more of the single antigen binding domains of the SDAB molecule are fused as gene fusions or polypeptide fusions with or without a linking group. The linking group can be any linking group apparent to those of skill in the art. For example, the linking group can be a biocompatible polymer 1 to 100 atoms long. In one embodiment, the linking group comprises or consists of a polyglycine residue, a polyserine residue, a polylysine residue, a polyglutamic acid residue, a polyisoleucine residue or a polyarginine residue, or a combination thereof. For example, a polyglycine linker or polyserine linker includes at least 5, 7, 8, 9, 10, 12, 15, 20, 30, 35 and 40 glycine residues and A serine residue may be included. Examples of linkers that can be used include Gly-Ser repeats with one, two, three, four, five, six, seven or more repeats, eg (Gly) ₄ -Ser (SEQ ID NO: 8) repeats. In some embodiments, the linker has the following sequence: (Gly) ₄ -Ser- (Gly) ₃ -Ser (SEQ ID NO: 9) or ((Gly) ₄ -Ser) n (SEQ ID NO: 10) And n is 4, 5 or 6.

  In one exemplary embodiment, a single chain polypeptide fusion of two single domain antibody molecules (eg, two camelid variable regions) that bind to a target antigen, eg, tumor necrosis factor alpha (TNFα), and Antigen-binding polypeptides composed of branched PEG molecules have been shown to have a dose-dependent therapeutic effect on already established arthritis in a transgenic mouse model. SDAB-01 is a humanized bivalent bispecific TNFα inhibitory fusion protein. The antigen for this protein is tumor necrosis factor-alpha (TNFα).

The complete amino acid sequence of the SDAB-01 polypeptide chain predicted from the DNA sequence of the corresponding expression vector is shown in FIG. 1 (residues are counted from the NH ₂ terminus as residue number 1 of SEQ ID NO: 1). Last amino acid residue encoded by the DNA sequence is ^{C 264,} constituting the COOH terminus of the protein. The predicted molecular weight of disulfide bonded SDAB-01 (without post-translational modification) is about 27000 Da. The molecular weight observed in the major isoform by nanoelectrospray ionization quadrupole time-of-flight mass spectrometry corresponds to 67000 Da, confirming the absence of post-translational modifications. The unique biochemical characteristics are as follows: 264 amino acids, molecular weight of 27365 Da, PI = 8.67 and UV = Ec = 2803 at 1.83.

  In FIG. 1, the complementarity determining region (CDR) is shown in bold. Amino acid linkers connecting these binding domains are shown in lower case.

Preparation of SDAB molecule An SDAB molecule can be recombinant, CDR grafted, humanized, camelized, deimmunized, and / or generated in vitro (eg, selected by phage display). It can consist of domain molecules. Techniques for generating antibodies and SDAB molecules and modifying them recombinantly are known in the art and are described in detail below.

  Many methods known to those skilled in the art are available to obtain antibodies. For example, monoclonal antibodies can be made by generating hybridomas according to known methods. The hybridomas thus formed are then screened using standard methods such as enzyme immunoassay (ELISA) and surface plasmon resonance (BIACORE ™) analysis to specifically bind to a particular antigen. Identify one or more hybridomas that produce the SDAB molecule. Any form of a particular antigen can be used as an immunogen, such as a recombinant antigen, a naturally occurring form thereof, any variant or fragment thereof, and an antigenic peptide thereof.

  One exemplary method of generating antibodies and SDAB molecules involves screening protein expression libraries, such as phage display libraries or ribosome display libraries. Phage display is described, for example, in US Pat. No. 5,223,409 of Ladner et al., Smith (1985) Science 228: 1315-1317, WO 92/18619, WO 91/17271, WO 91/17271. No. 92/20791, International Publication No. 92/15679, International Publication No. 93/01288, International Publication No. 92/01047, International Publication No. 92/09690, and International Publication No. 90/02809.

  In addition to the use of display libraries, specific antigens can be used to immunize non-human animals such as rodents such as mice, hamsters or rats. In one embodiment, the non-human animal includes at least a portion of a human immunoglobulin gene. For example, a large fragment of the human Ig locus can be used to engineer a mouse strain that is deficient in mouse antibody production. Hybridoma technology can be used to generate and select antigen-specific monoclonal antibodies derived from genes with the desired specificity. For example, XENOMOUSE ™, Green et al. (1994) Nature Genetics 7: 13-21, US Patent Application Publication No. 2003-0070185, International Publication No. WO 96/34096, published October 31, 1996. And International Application No. PCT / US96 / 05928 filed on April 29, 1996.

  In another embodiment, obtaining an SDAB molecule from a non-human animal and then using a recombinant DNA technique known in the art to produce a modified, eg humanized, deimmunized, chimeric SDAB molecule. Can do. Various approaches have been described for making chimeric antibodies and chimeric SDAB molecules. For example, Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851, 1985, Takeda et al., Nature 314: 452, 1985, U.S. Pat. No. 4,816,567 to Cabilly et al., Boss et See al., U.S. Pat. No. 4,816,397, Tanaguchi et al., European Patent No. 171496, European Patent No. 0173494, British Patent No. 2177096. Humanized antibodies and humanized SDAB molecules may be used, for example, in transgenic mice that express human heavy and light chain genes but are unable to express endogenous mouse immunoglobulin heavy and light chain genes. Can be produced. Winter describes an exemplary CDR grafting method that can be used to prepare the humanized antibodies and humanized SDAB molecules described herein (US Pat. No. 5,225,539). . All CDRs of a particular human antibody can be replaced with at least a portion of a non-human CDR, or only a portion of a CDR can be replaced with a non-human CDR. It is only necessary to replace the number of CDRs required for binding of a humanized antibody and SDAB molecule to a given antigen.

  Humanized antibodies can be generated by replacing sequences of the Fv variable domain that are not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Examples of methods for producing humanized antibodies or fragments thereof include Morrison (1985) Science 229: 1202-1207, Oi etal. (1986) BioTechniques 4: 214, and US Pat. No. 5,585,089, US Pat. No. 5,693,761, US Pat. No. 5,693,762, US Pat. No. 5,859,205 and US Pat. No. 6,407,213. These methods include isolating, manipulating, and expressing a nucleic acid sequence encoding all or part of an immunoglobulin Fv variable domain derived from at least one of a heavy or light chain. Such nucleic acids can be obtained from hybridomas that produce SDAB molecules for a given target, as described above, and from other sources. The recombinant DNA encoding the humanized SDAB molecule can then be cloned into an appropriate expression vector.

  In certain embodiments, humanized SDAB molecules are optimized for the introduction of conservative substitutions, consensus sequence substitutions, germline substitutions and / or backmutations. Such mutated immunoglobulin molecules can be generated by any of several techniques known in the art (eg, Teng et al., Proc. Natl. Acad. Sci. USA, 80: 7308-7312, 1983, Kozbor et al., Immunology Today, 4: 7279, 1983, Olsson et al., Meth. Enzymol., 92: 3-16, 1982), PCT International Publication No. 92/06193 or European Patent No. 0239400) Can be made according to the teachings of

  Techniques for humanization of SDAB molecules are disclosed in WO 06/122786.

SDAB molecules can also be modified by specific deletion of human T cell epitopes or “deimmunization” by methods disclosed in WO 98/52976 and WO 00/34317. In short, the heavy and light chain variable domains of the SDAB molecule can be analyzed for peptides that bind to MHC class II, and these peptides represent potential T cell epitopes (WO 98/52976 and International As specified in Publication No. 00/34317). For the detection of potential T cell epitopes, a computer modeling approach called “peptide threading” can be applied, as well as described in WO 98/52976 and WO 00/34317. In addition, motifs present in _VH and _VL sequences can be searched from a database of human MHC class II binding peptides. These motifs bind to any of the 18 major MHC class II DR allotypes and thus constitute potential T cell epitopes. Potential T cell epitopes to be detected can be eliminated by substituting a few amino acid residues in the variable domain or by a single amino acid substitution. Typically, conservative substitutions are made. In many cases, but not exclusively, amino acids common to positions in human germline antibody sequences can be used. Human germline sequences are described, for example, in Tomlinson, et al. (1992) J. Mol. Biol. 227: 776-798, Cook, GP et al. (1995) Immunol. Today Vol. 16 (5): 237-242 Chothia, D. et al. (1992) J. Mol. Biol. 227: 799-817, and Tomlinson et al. (1995) EMBO J. 14: 4628-4638. The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (summarized by Tomlinson, IA et al. MRC Center for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequences, for example for framework regions and CDRs. Human consensus framework regions can also be used, for example, as described in US Pat. No. 6,300,064.

Production of SDAB Molecules SDAB molecules can be produced by viable host cells that have been genetically engineered to produce proteins. Methods for genetically engineering cells to produce proteins are known in the art. See, for example, Ausabel et al., Eds. (1990), Current Protocols in Molecular Biology (Wiley, New York). Such a method involves introducing into a living host cell a nucleic acid that encodes the protein and allows expression of the protein. These host cells can be bacterial cells, fungal cells or animal cells grown in culture. Bacterial host cells include, but are not limited to, E. coli cells. Examples of suitable E. coli strains include HB101, DH5a, GM2929, JM109, KW251, NM538, NM539, and any E. coli strain that does not cleave foreign DNA. Fungal host cells that can be used include, but are not limited to, Saccharomyces cerevisiae, Pichiapastoris and Aspergillus cells. Some examples of animal cell lines that can be used are CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3 and WI38. New animal cell lines can be established (eg, by transformation, viral infection, and / or selection) using methods known to those skilled in the art. Optionally, the protein can be secreted into the medium by the host cell.

  In some embodiments, SDAB molecules can be made in bacterial cells, such as E. coli cells. For example, if the Fab is sequence-encoded in a phage display vector that includes an inhibitory stop codon between the display entity and the bacteriophage protein (or fragment thereof), the nucleic acid of the vector cannot be suppressed. It can be introduced into bacterial cells. In this case, the Fab is not fused to the gene III protein and is secreted into the periplasm and / or medium.

  SDAB molecules can also be made in eukaryotic cells. In one embodiment, the antibody (eg, scFv) is administered from Pichia (eg, see Powers et al. (2001) J Immunol Methods. 251: 123-35), Hansenula, or Saccharomyces. Expressed in yeast cells.

In one embodiment, the SDAB molecule is made in a mammalian cell. Exemplary mammalian host cells for expressing cloned antibodies or antigen-binding fragments thereof include Chinese hamster ovary cells (CHO cells) (see, eg, Kaufman and Sharp (1982) Mol. Biol. 159: 601-621). (Including dhfr ^- CHO cells described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220), lymphocyte cell lines such as NS0 myeloma cells and Examples include SP2 cells, COS cells, and cells derived from transgenic animals, such as transgenic mammals. For example, the cell is a breast epithelial cell.

  In addition to the nucleic acid sequence encoding the SDAB molecule, the recombinant expression vector may carry additional sequences, such as a sequence that regulates replication of the vector in a host cell (eg, an origin of replication) and a selectable marker gene. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, eg, US Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). See). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.

In an exemplary system for recombinant expression of SDAB molecules, recombinant expression vectors encoding both antibody heavy and light chains are introduced into dhfr ^- CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the heavy and light chain genes of the antibody are each derived from an enhancer / promoter regulator (eg, SV40, CMV, adenovirus, etc., eg, CMV enhancer / AdMLP promoter regulator or SV40 enhancer). / AdMLP promoter regulator) and operably linked to induce high transcription levels of the gene. The recombinant expression vector also carries the DHFR gene, which allows selection of CHO cells transfected with the vector using methotrexate selection / amplification. The selected transformed host cells can be cultured to express the antibody heavy and light chains, and intact antibody is recovered from the culture medium. Using standard molecular biology techniques, recombinant expression vectors are prepared, host cells are transfected, transformants are selected, host cells are cultured, and antibody molecules are recovered from the culture medium. be able to. For example, some SDAB molecules can be isolated by affinity chromatography.

  SDAB molecules can also be made by transgenic animals. For example, US Pat. No. 5,849,992 describes a method for expressing an antibody in the mammary gland of a transgenic mammal. A transgene is constructed that includes a milk-specific promoter, a nucleic acid encoding the antibody molecule, and a signal sequence for secretion. The milk produced by such transgenic mammal females has the antibody of interest secreted therein. Antibody molecules can be purified from milk or used directly depending on the application.

The binding properties of the SDAB molecule can be determined by any method, such as the following method: BIACORE ™
It can be measured by one of analysis, enzyme immunoassay (ELISA), x-ray crystal structure analysis, sequence analysis and scanning mutagenesis.

  The binding interaction between the SDAB molecule and the target (eg, TNFα) can be analyzed using surface plasmon resonance (SPR). SPR or biomolecular interaction analysis (BIA) detects biospecific interactions in real time without labeling any interactors. A change in mass at the binding surface of the BIA chip (which is an indicator of a binding event) causes a change in the refractive index of light near the surface. A detectable signal is generated by a change in refractive index measured as an indicator of real-time reaction between biomolecules. Methods using SPR are described, for example, in US Pat. No. 5,641,640, Raether (1988) Surface Plasmons Springer Verlag, Sjolander and Urbaniczky (1991) Anal. Chem. 63: 2338-2345, Szabo et al. (1995). Curr. Opin. Struct. Biol. 5: 699-705, and in online resources provided by BIAcore International AB (Uppsala, Sweden).

Information from the SPR can be used to provide an accurate and quantitative measure of the equilibrium dissociation constant (K _d ) and the kinetic parameters (including K _on and K _off ) for the binding of the molecule to the target. Such data can be used to compare different molecules. Information from the SPR can be used to develop the relationship between structure and activity (SAR). For example, kinetic parameters and equilibrium binding parameters of different antibody molecules can be evaluated. Mutant amino acids at a given position can be identified that correlate with specific binding parameters such as high affinity and slow K _off . This information can be combined with structural modeling (eg, using homology modeling, energy minimization, or x-ray crystal structure analysis or NMR structure determination). As a result, an understanding of the physical interaction between a protein and its target can be systematically described and used to guide other design processes.

Modified SDAB molecule An SDAB molecule may have an amino acid sequence that differs from the amino acid sequence of a naturally occurring domain, such as a VH domain, in at least one of the framework regions.

  The amino acid sequence of some SDAB molecules, eg, humanized SDAB molecules, is such that at least one amino acid position in at least one of the framework regions is the amino acid sequence of a naturally occurring domain, eg, a naturally occurring VHI-I domain. It should be understood that can be different.

  The invention also includes blends of derivatives of SDAB molecules. Such derivatives are generally modified, in particular by one or more chemical and / or biological (eg enzymatic) modifications of the SDAB molecule and / or the amino acid residues forming the SDAB molecule disclosed in the present invention. Can be obtained.

  Examples of such modifications, as well as amino acid residues within the SDAB molecule sequence that can be modified in this way (ie amino acid residues on the protein backbone or side chain), methods that can be used to introduce such modifications Examples of techniques and techniques, and potential uses and advantages of such modifications will be apparent to those skilled in the art.

  For example, such modifications may include the introduction of one or more functional groups, residues or moieties to or on the SDAB molecule (eg, by covalent linkage or in any other suitable manner) In particular, it may include the introduction of one or more functional groups, residues or moieties into the SDAB molecule that confer one or more desired properties or functional groups. Examples of such functional groups will be apparent to those skilled in the art.

  For example, such modifications increase the half-life, solubility and / or absorption of the SDAB molecule, reduce the immunogenicity and / or toxicity of the SDAB molecule, eliminate or reduce any undesirable side effects of the SDAB molecule, And / or one or more functional groups (e.g., providing other beneficial properties to the SDAB molecule and / or reducing undesirable properties of the SDAB molecule, or providing any combination of two or more of the above) (e.g. Introduction (covalently or in any other suitable manner) may be included. Examples of such functional groups and techniques for introducing them will be apparent to those skilled in the art and are generally all functional groups and techniques referred to in the general background art cited above, and in themselves modification of pharmaceutical proteins, In particular, it may include functional groups and techniques known for the modification of antibodies or antibody fragments (including ScFv antibodies and 148 single domain antibodies). See, for example, Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA (1980). Such functional groups can be linked, for example, directly (eg, covalently) to an SDAB molecule characterized in the present invention, or optionally via a suitable linker or spacer, as will also be apparent to those skilled in the art. it can.

Non-peptide linkers In the SDAB molecules described herein, one or more SDAB molecules and / or proteins and one or more acceptable polymers can be directly linked to each other and / or 1 It can be linked to each other via one or more suitable linkers.

  Certain terms are defined herein.

  The term “alkoxyl” or “alkoxy” as used herein refers to an alkyl group, as defined below, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like.

  The term “alkyl” refers to a radical of a saturated aliphatic group including straight chain alkyl groups and branched chain alkyl groups. In preferred embodiments, the straight chain alkyl or branched chain alkyl is 12 or less, unless otherwise specified, for example 1-12, 1-8, 1-6, or 1 It has ˜4 carbon atoms in its skeleton. Examples of alkyl moieties include methyl, ethyl, propyl (eg isopropyl), butyl (eg isobutyl or t-butyl).

The term “alkylene” refers to a divalent alkyl such as —CH ₂ —, —CH ₂ CH ₂ — and —CH ₂ CH ₂ CH ₂ —.

  The term “halo” or “halogen” refers to any radical of fluorine, chlorine, bromine or iodine.

In one embodiment, the linker moiety used to “link” a suitable acceptable polymer to the SDAB molecule described herein is represented by the moiety of formula (I):

In some embodiments, the linker is represented by the following formula:

  When two or more linkers are used in the SDAB molecules described herein, these linkers may be the same or different. Those skilled in the art will recognize and understand the most suitable linkers for use with the SDAB molecules of the present invention.

PEGylation One widely used technique for increasing the half-life of pharmaceutical proteins and / or reducing immunogenicity is to use suitable pharmacologically acceptable polymers such as poly (ethylene glycol) ( PEG) or derivatives thereof (eg methoxypoly (ethylene glycol) or mPEG). In general, using any suitable form of PEGylation, such as PEGylation used in the art for antibodies and antibody fragments, including but not limited to (single) domain antibodies and ScFv Can do. For example, Chapman, Nat. Biotechnol., 54, 531-545 (2002), Veronese and Harris, Adv. Drug Deliv. Rev. 54, 453-456 (2003), Harris and Chess, Nat Rev. Drug. Discov., 2, (2003), and WO 04/060965. Various reagents for PEGylation of proteins are also commercially available, for example from NOF America Corporation (eg for PEG formula B). Typically, site-specific PEGylation is used in particular via cysteine residues (see, eg, Yang et al., Protein Engineering, 16, 10, 761-770 (2003)). For example, for this purpose, PEG can be attached to a naturally occurring cysteine residue in the SDAB molecule, and the SDAB molecule is modified so that one or more cysteine residues are preferably introduced for attachment of the PEG. can do. Further, either using protein engineering techniques, the SDAB molecules described herein can be modified to suitably introduce one or more cysteine residues for PEGylation, Alternatively, an amino acid sequence containing one or more cysteine residues for PEGylation can be fused to the N-terminus and / or C-terminus of the SDAB molecule of the present invention.

  In connection with PEGylation, the present invention generally relates to the above PEGylation (1) increasing in vivo half-life, (2) reducing immunogenicity, and (3) known per se for PEGylation (4) essentially does not affect the affinity of the SDAB molecule (determined by a suitable assay as described in the examples below, for example). Does not reduce the affinity above 90%, above 50%, or above 10%), and / or (4) affects any other desired properties of the SDAB molecule It should be noted that any SDAB molecule that is PEGylated at one or more amino acid positions is also included, as not given. It will be apparent to those skilled in the art how to attach suitable PEG groups and suitable PEG groups specifically or non-specifically.

  The PEG used for the SDAB molecule and the proteins described herein may have a molecular weight of 1 KDa or more, such as 10 KDa or more and less than 200 KDa, such as less than 90 KDa. In some embodiments, the PEG used for the SDAB molecule and the proteins described herein can have a molecular weight in the range of 1 KDa to 100 KDa. Typically, SDAB molecules use PEG having a molecular weight in the range of greater than 5000, such as greater than 10,000, and less than 200000, such as less than 100,000, such as 20000-80000. In some embodiments, the PEG used for the SDAB molecule and the proteins described herein can have a molecular weight in the range of 10 KDa to 50 KDa. In some embodiments, the PEG used in the SDAB molecule and the proteins described herein can have a molecular weight in the range of 15 KDa to 45 KDa. In some embodiments, the PEG used for the SDAB molecule and the proteins described herein may have a molecular weight of 20 KDa. In some embodiments, the PEG used in the SDAB molecule and the proteins described herein can have a molecular weight of 40 KDa. In some embodiments, the PEG used in the SDAB molecule and the proteins described herein can have a molecular weight of 10 KDa.

  In some embodiments, each PEG molecule is independently a PEG monomer, polymer, or derivative thereof. In some embodiments, each PEG is a methoxy PEG derivative (mPEG) monomer, polymer, or derivative thereof. In some embodiments, each PEG molecule independently has a molecular weight of 1 KDa to 100 KDa. In some embodiments, each PEG molecule independently has a molecular weight of 10 KDa to 50 KDa. In some embodiments, each PEG molecule independently has a molecular weight of 40 KDa. In some embodiments, each PEG molecule independently has a molecular weight of 15 KDa to 35 KDa. In some embodiments, each PEG molecule independently has a molecular weight of 30 KDa. In some embodiments, each PEG molecule independently has a molecular weight of 20 KDa. In some embodiments, each PEG molecule independently has a molecular weight of 17.5 KDa. In some embodiments, each PEG molecule independently has a molecular weight of 12.5 KDa. In some embodiments, each PEG molecule independently has a molecular weight of 10 KDa. In some embodiments, each PEG molecule has a molecular weight of 7.5 KDa. In some embodiments, each PEG molecule independently has a molecular weight of 5 KDa.

  Another usually less typical modification is N-linked or O-linked glycosylation, usually as part of a post-translational modification and / or post-translational modification, depending on the host cell used to express the SDAB molecule. Including

In some embodiments, the PEG molecule is branched. In some embodiments, the PEG molecule is selected from moieties of formula (a) to formula (h):

(Where each PEG molecule is independently a PEG monomer, polymer, or derivative thereof). In some embodiments, each PEG molecule is an mPEG monomer, polymer, or derivative thereof. In some embodiments, the modified SDAB molecule comprises a linker of formula (I) linked to a PEG molecule and has a structure selected from:

(Where each PEG molecule is independently a PEG monomer, polymer, or derivative thereof). In some embodiments, each PEG molecule is an mPEG monomer, polymer, or derivative thereof.

In some embodiments, the modified SDAB molecule comprises a linker of formula (I) linked to a PEG molecule and has a structure selected from:
(Where each PEG molecule is independently a PEG monomer, polymer, or derivative thereof). In some embodiments, each PEG molecule is an mPEG monomer, polymer, or derivative thereof.

In some embodiments, the linker of formula (I) is linked to a PEG molecule represented by the following formula:

In some embodiments, the linker of formula (I) is linked to a PEG molecule represented by the following formula:

In some embodiments, the linker of formula (I) is linked to a PEG molecule represented by the following formula:

The linker-PEG molecule can associate with (eg, associate with) the SDAB molecule, thereby forming a modified SDAB molecule. Single domain molecules of the SDAB molecule may be arranged from N-terminus to C-terminus in the following order: TNFα-linked single domain molecule-TNFα-linked single domain molecule-PEG molecule (eg, branched PEG molecule ). In one embodiment, the modified SDAB molecule is represented by the following formula:

In one embodiment, the modified SDAB molecule is represented by the following formula:

In one embodiment, the modified SDAB molecule is represented by the following formula:

One exemplary embodiment of a modified SDAB molecule is represented by the following formula:

  The reactive group of the SDAB molecule is typically attached via a nucleophilic moiety attached to the SDAB molecule. In some embodiments, the nucleophilic moiety is sulfur (eg, the sulfur of a cysteine residue). In other embodiments, the nucleophilic moiety is nitrogen (eg, the nitrogen of the terminal α-amino group or the nitrogen-containing amino acid side chain (eg, the ε-amino group of the lysine chain)). In other embodiments, the nucleophilic moiety is a C-terminal group. The reactive group of the SDAB molecule is generally attached via an electrophilic moiety attached to the linker. In some embodiments, the electrophilic moiety is a carbonyl group (eg, an activated ester or aldehyde). In some embodiments, the electrophilic moiety is a maleimide group.

Administration and Treatment Methods An SDAB molecule is administered to a subject (eg, a human subject) alone or in conjunction with a second agent, eg, a therapeutically or pharmacologically active second agent, and TNFα A related disorder, such as an inflammatory disorder or an autoimmune disorder, can be treated or prevented (eg, one or more symptoms associated with a TNFα-related disorder are reduced or ameliorated). The term “treating” is effective in improving a condition, symptom or parameter associated with the disorder, or in suppressing the progression of the disorder to a statistically significant extent or to a level that is detectable to those skilled in the art. Refers to administering therapy in any amount, manner and / or manner. When used in therapy, the therapy can ameliorate, cure, maintain, or reduce the duration of the disorder or condition in the subject. For therapeutic use, the subject may have partial or complete signs of symptoms. Typically, treatment improves a subject's disorder or condition to a degree detectable by a physician or prevents the disorder or condition from worsening. Effective amounts, methods or modes can vary and are tailored to the subject, depending strongly on the subject.

  As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, the term “subject” and “subjects” refers to animals, such as non-primates (eg, cows, pigs, horses, donkeys, goats, camels, cats, dogs, It refers to mammals including guinea pigs, rats, mice, sheep) and primates (eg monkeys such as cynomolgus monkeys, gorillas, chimpanzees and humans).

  Non-limiting examples of immune disorders that can be treated include autoimmune disorders such as arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, lupus-related arthritis or ankylosing spondylitis), Scleroderma, systemic lupus erythematosus, Sjogren's syndrome, vasculitis, multiple sclerosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczema-like dermatitis), myasthenia gravis, inflammatory bowel disease ( IBD), Crohn's disease, colitis, diabetes (type I); inflammatory conditions such as inflammatory conditions of the skin (eg psoriasis); acute inflammatory conditions (eg endotoxemia, sepsis and septicemia, toxic shock syndrome and Infectious diseases); include but are not limited to graft rejection and allergies. In one embodiment, the TNFα-related disorder is an arthropathy such as rheumatoid arthritis, juvenile rheumatoid arthritis (RA) (eg moderate to severe rheumatoid arthritis), osteoarthritis, psoriatic arthritis, ankylosing spondylitis, polyarticular Juvenile idiopathic arthritis (JIA); or a disorder selected from one or more of psoriasis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, and / or multiple sclerosis.

  In certain embodiments, the SDAB molecule (or combination) is administered in conjunction with a second therapeutic agent. For example, in a TNFα SDAB molecule, the second agent can be an anti-TNFα antibody or a TNFα binding fragment thereof. Here, the second TNFα antibody has a different epitope specificity than the TNFα-binding SDAB molecule of the formulation. Other non-limiting examples of agents that can be combined with TNFα-binding SDAB include, for example, cytokine inhibitors, growth factor inhibitors, immunosuppressive agents, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors , Cytotoxic drugs, and cytostatic drugs. In one embodiment, the additional agent is a standard treatment for arthritis, including non-steroidal anti-inflammatory drugs (NSAIDs); corticosteroids (including prednisolone, prednisone, cortisone and triamcinolone); and diseases Modified anti-rheumatic agents (DMARD) such as methotrexate, hydroxychloroquine (Plaquenil) and sulfasalazine, leflunomide (Arava ™), tumor necrosis factor inhibitors (etanercept (Embrel ™), infliximab ™ (Remicade ™) ( Including adalimumab (with or without methotrexate) and adalimumab (Humira ™), anti-CD20 antibodies (eg Rituxan ™), soluble interleukin-1 receptors, eg anakinra t), gold, minocycline (Minocin (R)), penicillamine, and cytotoxic agents (azathioprine, including cyclophosphamide and cyclosporine) include, but are not limited to. Such combination therapies can beneficially utilize lower doses of therapeutic treatment, thereby avoiding the toxicities or complications that can be associated with various monotherapy.

  SDAB molecules can be administered in the form of liquid solutions (eg, injection solutions and infusion solutions). Such compositions can be administered by a parenteral form (eg, subcutaneous injection, intraperitoneal injection or intramuscular injection) or by inhalation. The terms “parenteral administration” and “parenteral administration” as used herein refer to modes of administration other than enteral and topical administration, usually by injection, subcutaneous or intramuscular, and intravenous Includes injections and infusions within, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subepidermal, subcapsular, intrathecal, intrathecal, epidural and substernal. In one embodiment, the formulations described herein are administered subcutaneously.

  A pharmaceutical composition or formulation is sterilized and stable under the conditions of manufacture and storage. The pharmaceutical composition can also be tested to meet regulatory and industry standards for administration.

  The pharmaceutical composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high protein concentration. Sterile injectable solutions may be sterile filtered, as required, in combination with one or a combination of the ingredients listed herein in the required amount in an appropriate solvent as required. Can be prepared. Generally, dispersions are prepared by incorporating the agents described herein into a sterile vehicle that contains the basic dispersion medium and the other required components among those listed above. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Inclusion of agents that delay absorption, such as monostearate salts and gelatin, in the composition can provide sustained absorption of the injectable compositions.

Composition / Formulation The formulation of the SDAB molecule includes an SDAB molecule, a compound that can act as an anti-freezing agent, and a buffer. The pH of the formulation is generally between pH 5.5 and 7.0. In some embodiments, the formulation is stored as a liquid. In other embodiments, the formulation is prepared as a liquid and then stored, eg, by lyophilization or spray drying. The dried formulation can be used as a dry compound, for example, as an aerosol or powder, or reconstituted to its original concentration or another concentration using, for example, water, buffer, or other suitable liquid.

  The SDAB molecule purification process is designed such that SDAB molecules are introduced into formulations suitable for long-term storage as frozen liquids and subsequent freeze drying (eg freeze drying using histidine / sucrose formulations). The formulation is lyophilized using a specific concentration of protein. The lyophilized formulation is then reconstituted with a suitable diluent (eg, water) as needed to re-dissolve the original formulation components to the desired concentration, generally the same or higher compared to the concentration prior to lyophilization. Can be made.

  Reconstitute the lyophilized formulation, depending on the amount of water or diluent added to the lyophilizate compared to the volume of the freeze-dried liquid initially, and the original (ie, prior to lyophilization) concentration Can produce formulations having different concentrations. Suitable formulations can be identified by assaying one or more parameters of antibody integrity.

Articles of Manufacture This application also provides an article of manufacture comprising a formulation as described herein and comprising instructions for using the formulation.

  For administration to a subject, for example, a formulation used as a pharmaceutical must be sterile. This is accomplished using methods known in the art, for example by liquid blending, or filtration through sterile filtration membranes before or after lyophilization and reconstitution. Alternatively, if the structure is not damaged, the ingredients of the formulation can be sterilized by autoclaving and then combined with the components that are filter sterilized or radiation sterilized to make the formulation.

  The pharmaceutical formulation can be administered using a transdermal delivery device such as, for example, a syringe for subcutaneous injection or a syringe including a multi-chamber syringe. In one embodiment, the device is a prefilled syringe with or with a needle. In other embodiments, the device is a prefilled syringe without a needle. The needle may be packaged with a prefilled syringe. In one embodiment, the device is a self-injection device, such as a self-injection syringe. In another embodiment, the injection device is a pen injector. In yet another embodiment, the syringe is a fixed needle syringe, a luer lock syringe, or a luer slip syringe. Other suitable delivery devices include stents, catheters, microneedles and implantable controlled release devices. The composition can be administered intravenously using standard IV equipment, including IV tubings, with or without in-line filters, for example.

  In certain embodiments, the syringe is suitable for use with an autoinjector device. For example, the autoinjector device may include a single vial system, such as a pen injector device for solution delivery. Such devices include, for example, Becton Dickensen (Franklin Lakes, NJ), Ypsomed (Burgdorf, Switzerland (www.ypsomed.com)), Bioject (Portland, Oreg.), National Medical Products, Weston Medical (Peterborough, UK), Medi- Produced or developed by Ject Corp (Minneapolis, Minn.) And Zogenix, Inc (Emeryville, CA), BD Pens, BD Autojector ™, Humaject ™, NovoPen ™, BD ™ Pen, AutoPen (TM), OptiPen (TM), GenotropinPen (TM), Genonorm Pen (TM), Humatro Pen (TM), Reco-Pen (TM), Roferon Pen (TM), Biojector (TM), IJect (TM) Trademark), J-tip Needle-Free Injec Commercially available from manufacturers such as tor (TM), DosePro (TM), Medi-Ject (TM). A device that includes a widely accepted dual vial system includes a pen injector system, eg, HumatroPen ™, that reconstitutes a lyophilized drug in a cartridge for delivery of a reconstitution solution.

  The article of manufacture can include a container suitable for containing the formulation. Suitable containers include devices, bottles, vials, syringes, test tubes, nebulizers (eg, ultrasonic nebulizers or vibrating mesh nebulizers), intravenous solution bags, or inhalers (eg, metered dose inhalers (MDI) or dry powder inhalers) (DPI)), but is not limited thereto. The container may be made of any suitable material such as glass, metal, or plastic such as polycarbonate, polystyrene or polypropylene.

  Generally, a container is a container of material that does not absorb significant amounts of protein from the formulation and is not reactive with the components of the formulation.

  The article of manufacture described herein can further include packaging material. In addition to information about use or administration, the packaging material comprises information about the conditions under which the product can be used, for example as required by regulatory authorities. For example, the packaging material can be a method of injecting a prefilled syringe containing a formulation described herein, or a lyophilized formulation within a specified period of time, for example, 2 hours to 24 hours or more. Can be reconstituted with an aqueous diluent to give instructions to the patient on how to form a solution. The formulations claimed in the present invention are useful for human pharmaceutical product applications.

  In certain embodiments, the formulation can be administered as a nebulizer. Examples of nebulizers include, but are not limited to, jet nebulizers, ultrasonic nebulizers, and vibrating mesh nebulizers. These classes use different methods to make aerosols from liquids. In general, any aerosol generating device capable of maintaining protein integrity in these formulations is suitable for delivery of the formulations described herein.

  In other embodiments, the pharmaceutical composition can be administered using a medical device. For example, the pharmaceutical composition may be a needleless hypodermic injection device such as US Pat. Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413. No. 4,941,880, No. 4,790,824, or No. 4,596,556. Examples of known implants and modules include US Pat. No. 4,487,603 (discloses an implantable microinfusion pump that dispenses medication at a controlled rate), US Pat. No. 4,486,194 (skin Through US Pat. No. 4,447,233 (discloses a pharmaceutical infusion pump that delivers medication at a precise infusion rate), US Pat. No. 4,447,224 (discloses a variable flow implantable infusion device for continuous drug delivery), US Pat. No. 4,439,196 (discloses an osmotic drug delivery system with multiple chamber compartments. And U.S. Pat. No. 4,475,196 (disclosing osmotic drug delivery systems). The therapeutic composition may be in the form of a biodegradable or non-biodegradable sustained release formulation for subcutaneous or intramuscular administration. See, for example, U.S. Pat. Nos. 3,773,919 and 4,767,628, and PCT International Publication No. 94/15587. Continuous administration can also be achieved using an implantable pump or an external pump. Administration can also be carried out intermittently, for example by injection once a day, or continuously at low doses, for example in a sustained release formulation. Improvements can be made to the delivery device to optimize for administration of the SDAB molecule. For example, the syringe can be siliconized to an extent that is optimal for storage and delivery of SDAB molecules. Of course, many other such implants, delivery systems and modules are also known.

  The invention also features a device for administering a first agent and a second agent. The device can include, for example, one or more housings for storing pharmaceutical preparations and can be constructed to deliver unit doses of a first agent and a second agent. The first agent and the second agent can be stored in the same compartment or in separate compartments. For example, the device can be administered after combining the agents. It is also possible to use different devices to administer the first agent and the second agent.

  The following examples are set forth to aid the understanding of the present invention, but are in no way intended to limit the scope thereof and are not to be construed as such.

Example 1. Generation of anti-TNFα component and genetic manipulation of SDAB-01 Two SDAB-01 bivalent humanized SDAB polypeptides are linked via a 30 amino acid flexible linker consisting of 6 repeats of 4 glycines and 1 serine. It was constructed by gene fusion of the same TNFα antigen-binding domain (amino acids 1-115 of SEQ ID NO: 1). To prepare for site-specific PEGylation, the free cysteine was engineered at the C-terminus following the three glycine amino acid linkers (FIG. 1). Protein was produced in a CHO mammalian expression system and purified by protein A affinity capture. The C-terminal cysteine was then reduced by dithiothreitol treatment and reacted with the maleimide functional group of activated 2 × 20 kDa branched PEG (FIG. 2). The final product was further purified and characterized from free PEG and a small portion of non-PEGylated protein.

  SDAB-01 is thereby derivatized with maleimide with two identical humanized anti-TNFα-specific SDAB molecules having the amino acid sequence of amino acids 1-115 of SEQ ID NO: 1 separated by a 30 amino acid flexible linker. A gene fusion with a specifically PEGylated C-terminal cysteine site (2 × 20 PEG) with a 40 kDa (2 × 20 kDa) branched polyethylene glycol (FIG. 3). FIG. 4A shows a linear mPEG-maleimide and two branched mPEG-maleimides containing SDAB-01. FIG. 4B is a scan diagram comparing the sizes of SDAB-01 and [SEQ ID NO: 1] -PEG40.

  Analytical analysis showed that the PEGylation efficiency between linear 40K mPEG-maleimide SDAB and branched 40K mPEG-maleimide SDAB was comparable. Anti-TNFα SDAB molecules PEGylated with either linear 40K mPEG-maleimide or branched 40K mPEG-maleimide showed comparable bioactivity. The apparent charge and shape appear to be approximately the same between the two branched 40K mPEG-maleimide materials (Branched PEG Formula A and Branched PEG Formula B).

  The construction of SDAB-01 as a bivalent format of two identical TNFα antigen binding domains (amino acids 1-115 of SEQ ID NO: 1) via a length-optimized flexible linker was performed in a cell-based TNFα neutralization assay. Compared to its monovalent format, it improved its efficacy by about 50 times. Site-specific PEGylation of engineered C-terminal cysteines gave drug candidates the desired pharmacokinetic profile with increased in vivo half-life without affecting their potency.

Example 2 Characterization of binding of SDAB-01 to membrane-bound TNFα by flow cytometry SDAB-01 is a flow site that binds to a recombinant Chinese hamster ovary (CHO) cell line expressing human TNFα on its cell surface. It has been demonstrated by a metric. A 13 amino acid deletion was introduced into the human TNFα coding region by site-directed mutagenesis, reducing protein cleavage and causing release of TNFα into the medium. A stable CHO strain was generated using this construct. Expression of TNFα on the cell surface was demonstrated by flow cytometry using a specific anti-human TNFα antibody. The cell line pW2128 CHO-TNF-D13 is stained with SDAB-01, followed by secondary staining with a biotinylated anti-PEG antibody and then detected by tertiary staining with streptavidin-PE for cell surface binding. (FIG. 5).

Example 3: Affinity of SDAB-01 for human TNF or rhesus monkey TNF Detailed characterization of anti-TNFα SDAB-01 binding to human TNFα and rhesus monkey TNFα was performed on a Biacore instrument using surface plasmon resonance. Biotinylated SDAB-01 was captured on the surface of the streptavidin sensor chip and various concentrations of human TNFα or rhesus monkey TNFα were tested in this experiment. TNFα protein was injected, allowed to associate at 100 μL / min for 1.5 minutes, and allowed to break 20 apart. Rate constants and Kd were determined by global fit using the 1: 1 binding model of Biavaluation software v4.1. Data presented as rate constants are the mean and standard deviation from at least two independent experiments. Kd was calculated from the average of association and dissociation rates (on and off rates). The affinity of SDAB-01 for human TNFα or rhesus monkey TNFα is shown in Table 1.

Table 1. Affinity of SDAB-01 for human TNFα or rhesus monkey TNFα as determined by Biacore

Example 4: Characterization of SDAB-01 in a cell-based cytotoxicity assay SDAB compared to SDAB molecule control 4 and non-PEGylated SDAB molecule control 3 in an L929 cell-based cytotoxicity assay using human TNFα or rhesus monkey TNFα Assessment of the bioactivity of -01. The ability of SDAB-01 to neutralize the cytotoxicity of TNFα (0.5 ng / ml) was evaluated in a cell-based dose response assay. SDAB-01 and non-PEGylated TNFα SDAB molecule Control 3 were assayed in the same experiment. The dose response curve is shown in FIG. 6 and the EC50 results are summarized in Table 2.

Table 2. Bioactivity of SDAB Control 4, SDAB-01 and Non-PEGylated SDAB Control 3

  These results indicate that SDAB-01 is able to neutralize both human and rhesus monkey TNFα in an L929 cell-based assay. The results also show that PEGylation has no significant effect on the neutralizing activity of SDAB-01.

Example 5 FIG. Comparison of TNFα SDAB-01 binding kinetics with various species of TNFα The purpose of this study was to determine the binding kinetics between SDAB-01 and various species of TNFα including humans, rhesus monkeys, rats, mice and rabbits. The equilibrium dissociation constant was studied to understand how to compare the binding affinity between these different species that can be used in efficacy models, pharmacokinetic models and toxicology models. Using a Biacore instrument, the reaction rate of binding in real time was measured by surface plasmon resonance. The rate constant was measured directly and the equilibrium dissociation constant was derived from the binding rate using Biacore evaluation software v4.1.

  For evaluation of TNFα binding, SDAB-01 was immobilized on the sensor chip surface at a density of 60 RU to 75 RU. Human TNFα and rhesus monkey TNFα bound similarly to SDAB-01 with fast association rates and very slow dissociation rates (FIGS. 7a, 7b). Binding to SDAB-01 reached saturation, depending on the concentration of human TNFα and rhesus monkey TNFα. At the highest concentration, binding reached equilibrium. In contrast to the high-affinity binding of SDAB-01 to human TNFα and rhesus monkey TNFα, the binding of rat TNFα and mouse TNFα to SDAB-01 was negligible (FIGS. 7c and 7d). At the highest concentration tested, 100 nM, rat TNFα binding and mouse TNFα binding observed a very low signal for binding, reaching only a binding response of less than 5 RU (FIG. 7). The highest concentration tested, the apparent fast dissociation rate at up to 100 nM and the lack of saturation are indicators of weak binding. In rat TNFα or mouse TNFα, there was no saturation and the binding rate was too fast to measure, making it impossible to calculate the equilibrium dissociation constant. This suggests that rat TNFα and mouse TNFα bind very weakly to SDAB-01, although there is some negligible binding. No binding between rabbit TNFα and SDAB-01 was observed even at the highest concentration of 400 nM rabbit TNFα tested (FIG. 7). These data indicate that SDAB-01 binds to rhesus monkey TNFα as much as human TNFα, but does not engage TNFα ligand in mice, rats or rabbits.

  Association and dissociation rate constants between human TNFα and rhesus monkey TNFα binding to SDAB-01 were calculated from the binding shown in FIG. 7 using a 1: 1 binding model (Table 3). Human TNFα and rhesus monkey TNFα had very similar association and dissociation rates, with nearly identical Kd values of 19.5 + 4.17 pM and 34.1 + 7.23 pM, respectively.

Table 3. Binding affinity of SDAB-01 to human TNFα and rhesus monkey TNFα

Example 6 Complement-dependent cytotoxicity and lack of antibody-dependent cytotoxicity against SDAB-01 SDAB-01 exhibits high neutralizing potency against human TNFα and monkey TNFα. Both CDC and ADCC are Fc-mediated effector functions. CDC can occur when the first protein, C1q, binds to the CH2 domain of the Fc region on two or more IgG molecules in an alternative complement cascade. This triggers downstream complement pathway components and ultimately results in the formation of a membrane injury complex on the surface of the cell, lysing the cell. ADCC interacts with FNF regions of anti-TNFα antibodies bound to TNFα on the cell surface and FcγR expressed on immune effector cells such as NK cells, monocytes, macrophages and neutrophils. Can cause death. The purpose of this study was to test for CDC and ADCC activity with SDAB-01 and compare it to anti-TNFα antibody control 1, anti-TNFα antibody control 2, anti-TNFα antibody control 3. Antibody control 1 and antibody control 2 have human IgG1 Fc and can therefore have effector functions. Antibody control 3 and SDAB-01 lack the Fc region.

  This analysis demonstrated that SDAB-01 and antibody control 3 did not have any CDC and ADCC activities compared to antibody control 1 and antibody control 2 (FIGS. 30 and 31). The Fc region of the antibody is required for the molecule to mediate CDC and ADCC activity, and antibody control 3 and SDAB-01 lack the Fc region. Therefore, SDAB-01 may bind to and neutralize TNFα on the cell surface without causing effector functional activity that can be cytotoxic.

Example 7: Effect of SDAB-01 on neutrophil infiltration The purpose of these in vivo studies was to demonstrate the ability of various doses of SDAB-01 to reduce cell infiltration induced by recombinant human TNFα in a murine air sac model. It was to evaluate.

  Tessier et al. (Jour of Immunol. 159: 3595-3602, 1997) have previously shown that injection of TNFα into mouse air sac induces leukocyte accumulation in the air sac. SDAB-01 was designed to bind to TNFα and neutralize the effects of TNFα. To test whether SDAB-01 had an effect on cell accumulation in an in vivo model, after SDAB-01 was administered to mice, TNFα was injected into the air sac. Cells were collected from the air sac and counted differentially at 6 hours after TNFα administration.

  The cyst fluid was collected at the end of each experiment (6 hours after administration of TNFα), and the cell number was determined by Cell Dyne. The results of Experiment 1 are shown in FIGS.

  SDAB-01 administered at 0.18 mg / kg significantly reduced cell infiltration into the air sac induced by 10 ng recombinant human TNFα. Neutrophil accumulation was also significantly inhibited by 0.18 mg / kg SDAB-01. Lymphocyte and monocyte infiltration was a trace component cell infiltration at 6 hours and this cell infiltration was not affected by SDAB-01 in this study.

  Experiment 2 was performed using the same protocol as experiment 1. The results are shown in FIGS. The results were consistent with those observed in Experiment 1, but in this experiment neutrophil infiltration was significantly inhibited at both 0.18 mg / kg and 0.09 mg / kg doses of SDAB-01. It was. Total cell infiltration was significantly reduced with 0.09 mg / kg SDAB-01 alone, and no significant reduction was observed with monocyte or lymphocyte infiltration.

  In Experiment 3, SDAB-01 was administered at the same dose as previously performed. As shown in FIGS. 12 and 13, a significant reduction in total leukocyte infiltration was observed at the 0.09 mg / kg dose, and a significant reduction in neutrophil infiltration was observed at both doses of SDAB-01. Lymphocytes were significantly reduced at the 0.09 mg / kg dose, but not significantly in the 0.18 mg / kg group. There was no effect on monocyte infiltration at any dose tested.

  In summary, significant inhibition of neutrophil infiltration compared to the control group at both concentrations of SDAB-01, with the exception of one study that gave a non-significant positive trend at the 0.09 mg / kg dose. Was observed (Table 4).

Table 4: Summary of murine air sac experiment using SDAB-01

  Administration of doses as low as 0.09 mg / kg SDAB-01 significantly reduced cell and neutrophil infiltration induced by 10 ng recombinant human TNFα. At any dose tested, there was little effect on lymphocyte infiltration and monocyte infiltration. These data indicate that SDAB-01 can consistently block neutrophil infiltration caused by recombinant human TNFα stimulation.

Example 8: Efficacy of SDAB-01 in Tg197 human TNFα transgenic mouse model of arthritis The therapeutic effect of SDAB-01 was evaluated in a TNFα transgenic mouse model of rheumatoid arthritis. In this model, TNFα transgenic mice develop chronic polyarthritis with a 100% incidence at 4-7 weeks of age. This disease relies on overexpression of human TNFα. The effects of various treatment doses (10 mg / kg, 3 mg / kg, 1 mg / kg, 0.3 mg / kg, 0.1 mg / kg, 0.03 mg / kg) of SDAB-01 were studied in the treatment regimen. Animals were randomly assigned to groups when 100% of the mice showed signs of disease. After assignment to the group, treatment with SDAB-01, anti-TNFα antibody control 2, control antibody or vehicle was started and continued twice a week for 7 weeks. All animals were scored weekly and blind for visual signs of disease symptoms. At the end of the study, the hind limbs were removed, processed and evaluated microscopically for disease indicators.

  In Experiment 1, treatment with SDAB-01 at doses of 10 mg / kg, 3 mg / kg, and 1 mg / kg was significantly more effective in preventing further development of arthritis in a dose-dependent manner compared to the vehicle-treated group. showed that. Treatment with higher doses of SDAB-01 (10 mg / kg, 3 mg / kg, 1 mg / kg) showed a marked improvement in histopathological scores compared to both control groups. Therefore, the minimal therapeutic dose that showed improvement in arthritis compared to the control treatment group, evaluated clinically by microscopy, was 1 mg / kg SDAB-01.

  In Experiment 2, treatment with SDAB-01 at doses of 10 mg / kg, 3 mg / kg and 1 mg / kg showed regression in both clinical and histopathological scores, indicating a therapeutic effect on established arthritis. Therefore, the minimum therapeutic dose that showed improvement in arthritis compared to the control treatment group, clinically assessed by microscopy, was 1 mg / kg.

  In summary, anti-TNFα treatment with SDAB-01 showed a dose-dependent therapeutic effect on established arthritis, as evidenced by prevention of disease progression and regression in both clinical and histopathological scores . The result of this treatment was a direct result of specific antagonism against human TNFα, as the control antibody treatment recapitulated lesions revealed by vehicle treatment in the Tg197 mouse arthritis model.

Experimental design SDAB-01 and anti-tetanus toxin (control) antibodies were prepared with Pfizer by standard methods. Infliximab (Remicade (c), anti-TNFα antibody, lot number 7HD98016) was purchased from MedWorld Pharmacy (catalog number NDC 57894-030-01).

Male Tg197 mice with the same type of human TNF-globin hybrid transgene (maintained in a CBA x C57BL / 6 genetic background) were mated with (CBA x C57BL / 6) F1 females. Atypical transgenic offspring were used in this study. When 100% of mice showed signs of arthritis, all mice were randomly assigned to treatment groups. From the day the animal was assigned to the treatment group, PF-05230905, control antibody (anti-tetanus toxin antibody), infliximab, or vehicle control (10 mM L-histidine, 5% sucrose buffer, lot number C-51683) by intraperitoneal injection in mice. , D-20216) was started. The dose and frequency of administration are listed in each experimental subsection. Both hind limbs of each mouse were evaluated for disease progression at specified intervals as follows: 0 No arthritis (normal appearance and flexion).
0.5 Onset of arthritis (mild joint swelling).
1 Mild arthritis (joint distortion).
1.5 With the above-mentioned finger deformation, the strength against bending is weak.
2 Moderate arthritis (no strength against severe swelling, joint deformation, flexion).
2.5 Deformation of limb fingers as described above.
3 Severe arthritis (joint stiffness detected in flexion and severe movement disorders).

Each mouse was then assigned an average score of 0-3. To monitor disease progression, 4 littermates of Tg197 mice also suffering from arthritis were sacrificed at 6 weeks of age, the starting point of treatment. At the end of the study, all mice were sacrificed and histopathological analysis of the ankle joint was performed. The experimental mouse scores were compared to the scores of 4 litters. Histopathological scores were evaluated blindly by microscope at 0-4 as follows:
0 No detectable lesions 1 Presence of synovial hyperplasia and polymorphonuclear infiltration 2 Pannus and fibrous tissue formation and local subchondral bone erosion 3 Cartilage destruction and bone erosion 4 Extensive cartilage Fracture and bone erosion.

Experiment 1
In Experiment 1, the efficacy of various doses of SDAB-01 was evaluated in a therapeutic TNFα transgenic murine model of rheumatoid arthritis. Mice were monitored twice a week for signs of arthritis. If 100% of mice showed signs of disease, all animals were randomly assigned to treatment groups. Atypical Tg197 mice were divided into groups of 8 mice each. Twice a week SDAB-01 (10 mg / kg, 3 mg / kg, 1 mg / kg, 0.3 mg / kg, 0.1 mg / kg), control antibody (10 mg / kg), infliximab (10 mg / kg, 3 mg) / Kg) or vehicle (histidine / sucrose buffer, 10 mL / kg). Treatment continued for 7 weeks and gross changes in joint morphology (arthritic score) and average body weight of each animal were recorded weekly. After euthanization with CO2, serum was collected and the two hind limbs of each animal were processed for histological evaluation.

  In Experiment 1, SDAB-01 administration was significantly more effective in improving weight loss (FIG. 14) and suppressing disease progression (FIG. 15) compared to the vehicle-treated group. Infliximab was identical to SDAB-01 (10 mg / kg, 3 mg / kg, 1 mg / kg) administration in stabilizing clinical scores.

  The severity of the disease evaluated on the last day of scoring is shown in FIG. The number of animals with reduced disease symptoms was greatest in the group treated with SDAB-01 (10 mg / kg, 3 mg / kg, 1 mg / kg) and infliximab (10 mg / kg) compared to vehicle or control antibody .

  One section stained with hematoxylin and eosin from each of the two hind limbs of each mouse was evaluated blindly and microscopically. Treatment with SDAB-01 (10 mg / kg, 3 mg / kg, 1 mg / kg) for established arthritis has shown efficacy by suppressing disease progression and gradual regression of histopathological scores . The result of this treatment was a direct result of specific antagonism against human TNFα because the control antibody treatment reproduced the lesions revealed by vehicle treatment (FIGS. 17 and 18).

Experiment 2
In Experiment 2, the effect of treatment with 10 mg / kg, 3 mg / kg, 1 mg / kg, 0.3 mg / kg and 0.1 mg / kg of SDAB-01 was repeated twice a week, twice a week , 0.03 mg / kg SDAB-01, and twice a week, 10 mg / kg and 3 mg / kg infliximab. Administration of SDAB-01 (10 mg / kg, 3 mg / kg, 1 mg / kg, 0.3 mg / kg, 0.1 mg / kg and 0.03 mg / kg) showed significant effects by improving weight loss (FIG. 19). However, only 10 mg / kg, 3 mg / kg and 1 mg / kg doses succeeded in suppressing disease progression compared to the vehicle treated group (FIG. 20). Treatment with SDAB-01 (0.3 mg / kg) or infliximab (3 mg / kg) resulted in moderate but not significant improvement in clinical evaluation compared to either the vehicle-treated group or the control antibody-treated group. I was drowned.

  The severity of the disease evaluated on the last day of scoring is shown in FIG. The number of animals with reduced disease symptoms was greatest in the group treated with SDAB-01 (10 mg / kg, 3 mg / kg, 1 mg / kg) and infliximab (10 mg / kg) compared to vehicle control.

  One section stained with hematoxylin and eosin from each of the two hind limbs of each mouse was evaluated blindly and microscopically. Treatment with SDAB-01 (10 mg / kg, 3 mg / kg, 1 mg / kg) showed efficacy against arthritis established by reducing disease progression and gradual regression of histopathological scores (FIG. 22). The result of this treatment was a direct result of the specific antagonism against human TNFα since the human control antibody treatment reproduced the lesions revealed by the vehicle treatment. The three higher doses of SDAB-01 (10 mg / kg, 3 mg / kg and 1 mg / kg) were found to be both controls as there was no significant difference between the vehicle-treated group score and the control antibody-treated group score. It was effective as evidenced by a significant reduction in histopathological score compared to the group (FIG. 23). Therefore, the minimum effective dose evaluated clinically by microscopy was 1 mg / kg SDAB-01.

  Treatment with three higher doses of SDAB-01 (10 mg / kg, 3 mg / kg and 1 mg / kg) resulted in improved histopathology scores. These scores were significantly lower than those of control littermates collected at the start of the study.

  With 1 mg / kg MED, the average measured SDAB-01 concentration in steady state (end-bleed) was 4.81 μg / mL, which is a single 1 mg / kg dose to Tg197 mice. Compared to the 7.70 μg / mL steady-state (at the end of bleeding) serum concentration predicted based on the pharmacokinetic profile of SDAB-01 after IP administration, it was within a 2-fold difference. The average serum SDAB-01 concentration at steady state (at the end of bleeding) was 0.21 μg / mL, 42.1 μg / mL and 120 μg / mL in the 0.3 mg / kg, 3 mg / kg and 10 mg / kg dose groups, respectively. there were. In the 0.03 mg / kg and 0.1 mg / kg dose groups, all but one animal had a serum SDAB-01 concentration below the limit of quantification of 0.049 μg / mL.

Example 9 PEGylation of bivalent SDAB molecules expressed in Pichia pastoris Dithiothreitol (DTT) was added to the neutralized fraction to reduce potential disulfide bridges formed between carboxy-terminal cysteines of SDAB molecules. An overnight incubation of 10 mM final concentration of DTT and 4 ° C. was found to be optimal. Reduction was assessed by analytical size exclusion chromatography (SEC). Then 25 ml of reduced SDAB molecule was added to 75 ml Dulbecco's PBS (D-PBS) and injected onto a Sup75 10/300 GL column equilibrated with D-PBS.

  Non-reduced SDAB molecules and DTT were removed by preparative SEC on a Hiload 26/60 Superdex 75 preparative grade column equilibrated with D-PBS.

  The concentration of reduced SDAB molecules was determined by measuring absorbance at 280 nm. A Uvikon 943 Double Beam US / VIS spectrophotometer was used. Absorption was measured with a wavelength scan from 245 nm to 330 nm. Two precision cells made of Quartz Suprasil were used. First, the absorbance of the blank was measured at 280 nm by placing two cells filled with 900 μl D-PBS. Samples were diluted (1/10) by adding 100 μl of sample to the first cell and mixing it before reading. The absorbance of the sample was measured at 280 nm. The concentration was calculated with the following formula:

  To PEGylate SDAB molecules, a 5 × molar excess of freshly made 1 mM PEG40 solution was added to the reduced SDAB molecule solution.

  The SDAB molecule-PEG mixture was incubated for 1 hour at room temperature with gentle agitation and then transferred to 4 ° C. PEGylation was evaluated with analytical SEC. Then 25 μl of SDAB molecule was added to 75 μl of D-PBS and injected onto a Sup75HR 10/300 column equilibrated with D-PBS. PEGylated SDAB molecules were eluted in the column excluded volume range (greater than 75 KDa).

  PEGylated SDAB molecules and non-PEGylated SDAB molecules were separated by cation exchange chromatography (CEX-buffer A was 25 mM citric acid and buffer B was 1 M NaCl in PBS). The sample was diluted, the electrical conductivity was 5 mS / cm, and the pH was adjusted to 4.0. The column was equilibrated and washed thoroughly with buffer A after sample application. PEGylated SDAB molecules were eluted with a 3 CV gradient.

  For recovered SDAB molecules, the buffer was exchanged into D-PBS by SEC on a Hiload 26/60 Superdex 75 prep grade column equilibrated with D-PBS. The SDAB molecules were then made LPS free by passing through an anion exchange column. The column was sanitized with 1M NaOH and then equilibrated with endotoxin-free D-PBS.

Biotinylation To biotinylate the SDAB molecule, a 5 × molar excess of biotin in a 10 mM stock solution was added to the reduced SDAB molecule. The biotin SDAB molecule mixture was incubated for 1 hour at room temperature with gentle agitation and then stored at 4 ° C.

  The purity of the biotinylated SDAB molecule was controlled by analytical SEC. Subsequently, 25 μl of biotinylated SDAB molecule was added to 75 μl of D-PBS and injected onto a Sup75HR 10/300 column equilibrated with D-PBS. The resulting chromatogram shows that the SDAB molecule biotin does not need further purification, and no dimerization of the SDAB molecule due to oxidation of free sulfhydryls could be detected. The buffer was exchanged into D-PBS by passing through a Sephadex G25 fine column as a desalting column.

  The SDAB molecule-biotin was made LPS free by passing through an anion exchange column. The column was flushed overnight with 1M NaOH and then equilibrated with D-PBS.

Example 10a. Pharmacokinetics of SDAB-01 in male cynomolgus monkeys after single intravenous and subcutaneous administration In the first study, SDAB-01 was administered as a single IV bolus injection or SC at 3 mg / kg (based on protein content). Male cynomolgus monkeys (n = 3 per group: monkey SAN 1-3 for IV, monkey SAN 4-6 for SC) were administered by bolus injection. Serum samples were collected from each animal for PK analysis before administration (0 hours) and 0.083 hours to 1536 hours after administration. Additional serum samples were taken before administration (0 hours) and 336 hours, 672 hours, 1008 hours and 1536 hours after administration to evaluate the formation of anti-SDAB-01 antibodies. Serum SDAB-01 concentrations were determined using a qualified enzyme immunoassay (ELISA) and the results were used to determine pharmacokinetic parameters for SDAB-01. The presence of anti-SDAB-01 antibody was determined using a limited ELISA.

  The mean serum concentration-time profile of SDAB-01 in male cynomolgus monkeys after IV or SC administration is shown in FIG. The average pharmacokinetic parameters of SDAB-01 after IV or SC administration in monkeys are summarized in Table 5.

Table 5: SDAB-01 mean (± SD) in male cynomolgus monkeys (n = 3 per treatment group) after a single IV or SC dose of 3 mg / kg (based on protein content)
Pharmacokinetic parameters

SDAB-01 was well absorbed from the injection site after 3 mg / kg SC administration. After a single SC dose of 3 mg / kg in 3 male cynomolgus monkeys, a mean maximum serum concentration (C _max ) of 31.7 ± 2.72 μg / mL was observed at 72 hours post-dose, thereby allowing a post-SC injection. The absorption of SDAB-01 was shown to be a slow process. The terminal half-life in 3 monkeys ranged from 110 hours to 131 hours, with an average value of 123 hours (approximately 5 days). The relatively short t _1/2 observed after SC administration may be due to the formation of anti-SDAB-01 antibodies.

Two monkeys from the SC treated group that are positive for anti-SDAB-01 antibody. The average AUC of 3 monkeys was 8958 μg · hr / mL. Bioavailability after SC administration in monkeys cannot be accurately determined from this study due to the formation of anti-SDAB-01 antibodies in both IV and SC-treated monkeys. However, an estimate can be obtained using the AUC _0-∞ ratio between SC and IV administration, and this estimate was found to be approximately 69.3%. This value should be used with caution since it may be low or high for the SDAB-01 bioavailability in monkeys.

In summary, the formation of anti-SDAB-01 antibodies was detected in 50% (3/6) of the animals that received anti-SDAB-01. The incidence of anti-SDAB-01 antibodies was 33.3% (1/3) in animals in the 3 mg / kg group IV and 66.7% (2/3) in animals in the 3 mg / kg group SC
Met. Antibodies (log titers between 2.19 and 2.52) were detected at 1008 hours and 1536 hours after administration in monkey SAN1 (IV treatment group) and monkey 5 (SC treatment group). The antibody (log titer of 1.71) was detected 1536 hours after administration with monkey SAN4 (SC treated group). These animals were considered to have an immune response to SDAB-01 because all pre-dose samples were negative. Note that circulating levels of SDAB-01 may prevent detection of anti-SDAB-01 antibodies.

  The half-life of SDAB-01 is shorter in monkeys that were positive for anti-SDAB-01 antibody formation, so that formation of anti-SDAB-01 antibodies may affect SDAB-01 pharmacokinetics in monkeys It was suggested.

In the second study, male and female cynomolgus monkeys (n = 12 per group) received a single 5 mg / kg IV dose, 100 mg / kg IV dose and 100 mg / kg SC dose SDAB-01. Serum concentrations were then measured using a limited ELISA. After IV administration of SDAB-01 at 5 mg / kg or 100 mg / kg, the mean AUC _0-∞ , CL and t _1/2 values were 24600 μg · h / mL and 395000 μg · h / mL, 0.210 mL / hr / kg and 0.263 mL / hr / kg, and 149 hours and 144 hours. Systemic exposure (Cmax, AUC _0-∞ and AUC _0-168 ) increased approximately in proportion to the dose as the dose was increased. After a single 100 mg / kg SC administration, mean Tmax, AUC _0-∞ and t _1/2 values were 150 hours, 352000 μg · h / mL and 165 hours, respectively. The bioavailability after SC administration (estimated using 100 mg / kg IV administration and mean AUC _0-∞ values after SC administration) was 89%. The incidence of anti-SDAB-01 antibody was 4 out of 12 animals (33.3%) in the 5 mg / kg (IV) dose group, 100 mg / kg (IV) dose group, and 100 mg / kg (SC) dose group, respectively. ), 1 out of 12 animals (8.3%), and 1 out of 12 animals (8.3%).

Example 10b. Comparison of the serum pharmacokinetics of SDAB-01 (TNFα SDAB molecule 2 × 20 PEG), TNFα SDAB molecule 4 × 10 PEG, and linear 1 × 40 PEG of TNFα SDAB molecule TNFα SDAB molecule branched 2 × 20 kDa PEG construct, TNFα SDAB molecule B6CBAF1 / J mice after a single IV dose of 2 mg / kg or 3 mg / kg (based on protein content) of serum PK profile of branched 4 × 10 kDa PEG construct and linear 1 × 40 kDa PEG construct of TNFα SDAB molecule, Sprague Dawley rats and cynomolgus monkeys were examined. Serum concentrations were determined using either specific ELISA (mouse and monkey) or gamma ray measurement (rat).

  In all three tested, the branched 2 × 20 kDa PEG construct had significantly higher exposure (AUC) compared to the linear 1 × 40 kDa PEG construct (p <0.05) (FIG. 25 and Table 6 to Table 8). In particular, the relative increase in AUC0-∞ normalized by the average dose for the branched 2 × 20 kDa PEG construct compared to the linear 1 × 40 kDa PEG construct is about 94%, 102% and 136 for mouse, rat and monkey, respectively. %Met. Thus, the total body clearance (CL) of the branched 2 × 20 kDa PEG construct is lower compared to the linear 1 × 40 kDa PEG construct, and the elimination half-life (t1 / 2) of the branched 2 × 20 kDa PEG is It was considered longer compared to the x40 kDa PEG construct. In particular, the relative reduction in mean CL value for the branched 2 × 20 kDa PEG construct is about 48%, 50% and 66% in mice, rats and monkeys, respectively, and the relative increase in mean t1 / 2 values is It was 43%, 26%, and 54% in rats and monkeys, respectively.

  In rats and monkeys, the branched 4 × 10 kDa PEG construct also had higher mean serum AUC0-∞ and lower CL compared to the linear 1 × 40 kDa PEG construct, but not in mice (Table). 6 to Table 8). In rats and monkeys, the degree of change in the PK parameter for the branched 4 × 10 kDa PEG construct compared to the linear construct is less pronounced compared to the degree of change in the PK parameter for the branched 2 × 20 kDa PEG construct. (43% -51% increase in AUC0-∞ and 35% -45% decrease in CL).

Table 6. Pharmacokinetic parameters of PEGylated TNFα SDAB molecules after a single IV administration to B6CBAF1 / J mice.

  Male B6CBAF1 / J mice received a single IV bolus dose of the specified test substance. Serum samples were taken from each mouse (n = 3 per time point) from 5 minutes to 14 days after a single dose, and serum concentrations were determined by specific ELISA. PK parameters were determined by non-compartmental analysis using sparse sampling, and statistical analysis of AUClast / dose values was performed by Dunnett's post-test using ANOVA. A linear 1 × 40 PEG group was used as a control.

An asterisk ( ^* ) indicates a statistically significant difference (p <0.05) compared to the linear PEG group.
C5 min = concentration at 5 min, the first sampling time after IV administration.
CL = total body clearance based on serum concentration.
Vdss = distributed volume in steady state.
t1 / 2 = elimination half-life.
AUC0-∞ = area under the concentration time curve from time 0 to infinity.
AUClast = time 0—area under the concentration time curve at the sampling time at which quantifiable concentrations are seen

Table 7. Pharmacokinetic parameters (average ± SD) of 125I-labeled PEGylated TNFα SDAB molecules after single IV administration to Sprague-Dawley rats

Male Sprague-Dawley rats are given a single IV bolus dose of the designated 125I-labeled test substance, and serum samples are taken from 5 minutes to 24 days after administration to determine the radioequivalent (RE) concentration in the serum. Determined by gamma ray measurement. PK parameters were calculated for each individual animal by non-compartmental analysis (n = 7 for 2 × 20 kDa PEG construct and 4 × 10 kDa PEG construct and n = 5 for 1 × 40 kDa PEG construct). Statistical analysis of AUC0-∞, AUC0-∞ / dose, CL, Vdss and t1 / 2 values was performed by Dunnett's post-test using ANOVA. A linear 1 × 40 kDa PEG group was used as a control. An asterisk ( ^* ) indicates a statistically significant difference (p <0.05) compared to linear PEG.

Table 8. Pharmacokinetic parameters of PEGylated TNFα SDAB molecules after single IV administration to cynomolgus monkeys (mean ± SD)

Male cynomolgus monkeys are administered a single IV bolus dose of the specified test substance and serum samples are collected with 2 × 20 kDa PEG, 4 × 10 kDa PEG and 1 × 40 kDa PEG constructs, 5 min-62 days, 57 Samples were collected on day and day 56 and serum concentrations were determined by ELISA. PK parameters were calculated for each individual animal by non-compartmental analysis (n = 3 per construct). Data points with a rapid concentration drop were not used for PK calculation (1 of 3 monkeys administered 2 × 20 kDa PEG construct). _AUC 0~∞, AUC 0~∞ / administration, CL, a statistical analysis of the values of _{Vd ss} and _{t 1/2,} was performed by Dunnett's post test using ANOVA. A linear 1 × 40 kDa PEG group was used as a control. An asterisk ( ^* ) indicates a statistically significant difference (p <0.05) compared to the linear PEG group.

  Further studies were performed with the SDAB-01 construct only:

  Initially, mouse and monkey serum samples were analyzed using two different immunoassay formats: whole molecule versus immunoassay that measures the protein portion of the molecule. The protein detection assay captured the PEGylated drug conjugate through the protein moiety by utilizing a biotinylated target molecule. The polyclonal anti-drug antibody detector also bound to the protein portion of the molecule, so the assay detected free protein and PEGylated protein. The whole molecule detection assay used the same capture mode as the Protein Detection assay, but detection occurred with the PEG moiety via a monoclonal rabbit anti-PEG antibody. This detection antibody is specific for the methoxy group of the PEG molecule. This assay format had little effect on the PK profile, and parameters were calculated in mouse and monkey animal models.

  Second, the pharmacokinetic profile of SDAB-01 was examined after a single SC or IP administration to mice. After a single 2 mg / kg SC or 3 mg / kg IP administration to male B6CBAF1 / J mice, the Tmax is 24 hours and the t1 / 2 values are 52.4 hours (approximately 2.2 days), respectively. ) And 57.7 hours (approximately 2.4 days). The bioavailability after IP administration or SC administration was 68.7% and 56.6%, respectively. After a single 0.3 mg / kg IP administration to male Tg197 mice, the values of Tmax, t1 / 2 and AUC0-∞ were 6 hours, 24.6 hours and 165 μg · h / mL, respectively. Increasing the IP dose to 1 mg / kg increased the exposure dose roughly in proportion to the dose (AUC0-∞ = 528 μg · h / mL), with Tmax (6 hours) and t1 / 2 (21.4 hours) The value was similar to that observed at 0.3 mg / kg.

Example 10c. Biodistribution of SDAB-01 (TNFα SDAB molecule 2 × 20 PEG) and linear 1 × 40 PEG of TNFα SDAB molecule Biological 2 × 20 kDa PEG construct of TNFα SDAB molecule and linear 1 × 40 kDa PEG construct of TNFα SDAB molecule Distribution was examined over 7 days (168 hours) in B6CBAF1 / J mice after a single IV administration of test substance labeled with 125 I at 0.3 mg / kg (based on protein content). Serum and tissue radioequivalent (RE) concentrations were determined using gamma counting, and serum and tissue exposure (AUC 0-168 hr) and tissue to serum (T / S) AUC ratio were calculated.

  Similar to the observations of previous studies in B6CBAF1 / J mice with non-radiolabeled PEG conjugates, the branched 2 × 20 kDa PEG construct has about 80% higher AUC 0-168 hr compared to the linear 1 × 40 kDa construct. (P <0.05) (FIG. 26). The branched construct also had significantly higher exposure in some but not all tissues tested (FIG. 26). In particular, the increase in AUC 0 to 168 hr for the branched 2 × 20 kDa PEG construct compared to the linear 1 × 40 kDa PEG construct is 72%, 115%, 43%, 55% in the heart, lung, muscle, skin and stomach, respectively. 80%. The T / S AUC ratio (Table 9) and the T / S concentration ratio (data not shown) were roughly similar between the two constructs for these tissues.

  For serum, heart, lung, muscle, skin and stomach, AUC0-168hr of fat, kidney, liver and spleen are comparable with the two constructs and lower T / S AUC ratio with the branched 2 × 20 kDa PEG construct (Table 9) and T / S concentration ratios (data not shown).

  With both TNFα SDAB molecule linear 1 × 40 PEG and SDAB-01, approximately 60% of total administered radioactivity is excreted in the urine within one week (168 hours) after administration, and the radioactivity excreted in the urine Most of the potency (approximately 70%) was attributed to free iodine.

Table 9. Tissue-to-serum (T / S) AUC ratio of ¹²⁵ I-labeled PEGylated TNFα Nanobody after a single 0.3 mg / kg IV dose to B6CBAF1 / J mice

B6CBAF1 / J mice were given a single 0.3 mg / kg IV of ¹²⁵ I-labeled TNFα SDAB molecule branched 2 × 20 kDa PEG (black bar) or TNFα SDAB molecule linear 40 kDa PEG (grey bar). A bolus was administered. Serum and tissue samples (n = 8-12 per time point) are collected over 7 days (168 hours) and radioequivalent (RE) concentrations in tissues and serum are gamma-measured as described herein Determined by. Serum AUC _{0-168 hr} (in μg × equivalent / mL) and each tissue AUC _{0-168 hr} (μg × equivalent / g) were determined by non-compartmental analysis using sparse sampling, and tissue versus serum (T / S) AUC ratio (AUC _{0-168 hr, tissue} / AUC _{0-168 hr, serum} ) was calculated.

Example 11 Biophysical analysis of SDAB and control molecules Further biophysical analysis was performed to investigate the potential cause for the different PK profiles of the three TNFα SDAB molecules 40 kDa PEG conjugates.

  CEX-HPLC was performed to monitor the charge heterogeneity of the three constructs. A representative chromatographic profile is shown in FIG. A significant amount of charge heterogeneity was observed with all PEGylated TNFα SDAB molecular conjugates. The main peak of the linear PEG conjugate elutes with a slower retention time when compared to the two branched conjugates (2 × 20 kDa and 4 × 10 kDa), so that the linear conjugate is compared to the branched conjugate. Suggesting that it has more positive charge on the surface. The main peak retention times for the two branched conjugates (2 × 20 kDa and 4 × 10 kDa) were similar. In comparison, unconjugated protein eluted much later than all tested PEGylated conjugates, indicating that the unconjugated protein had a greater positive surface charge density. The theoretical isoelectric point of the unconjugated protein is greater than 9, so this protein is expected to have a net positive charge in CEX running buffer at pH 4.0.

  Size distribution and mass distribution were determined using SE-HPLC with multi-angle light scattering (MALS), differential refractive index (dRI), and on-line quasi-elastic light scattering (QELS) monitored by UV absorbance. Since PEG in the TNFα SDAB molecule-PEG conjugate does not absorb at 280 nm, it is possible to determine the protein and PEG distribution in the conjugate using SEC-MALS with UV detection and dRI detection. The calculated protein and PEG mass distributions were consistent with each other for all three conjugates (Table 10 and Figure 28).

  The branched 4 × 10 kDa PEG conjugate has a significantly slower elution volume at SEC-MALS than the branched 2 × 20 kDa PEG conjugate and the linear 1 × 40 kDa PEG conjugate, thereby branching 4 × 10 kDa PEG It was shown that the conjugate was hydrodynamically smaller compared to the other two conjugates (FIG. 29). The smaller hydrodynamic radius (Rh, defined as the radius of a sphere with the same diffusion coefficient as the sample being measured) of the 4 × 10 kDa branched PEG conjugate was confirmed by QELS measurements (Table 10).

  Using the angular dependence of scattered light measured by MALS, the root mean square (RMS) radius distribution can be determined. The RMS radius (also referred to as the radius of rotation, also referred to as Rg) is the measurement of the root mean square distance from the center of mass of all parts of a molecule at any given time, and is the average that the molecule occupies Give information about the volume. Both the branched 2 × 20 kDa PEG conjugate and the branched 4 × 10 kDa PEG conjugate had a smaller Rg (RMS radius) than the linear PEG conjugate (Table 10 and FIG. 29).

  Finally, conformational information can be obtained by calculating the RMS / Rh (Rg / Rh) ratio: the larger the ratio value, the more the molecule will stretch or expand. The RMS / Rh ratio is 1.77, 1.45 and 1.37 for linear 1 × 40 kDa PEG conjugate, branched 2 × 20 kDa PEG conjugate and branched 4 × 10 kDa PEG conjugate, respectively, thereby linear Conjugates with 1 × 40 kDa PEG were shown to have an extended conformation than smaller conjugates containing branched PEG (Table 10). Note that the SE-HPLC method used to analyze PEGylated conjugates is not suitable for side-by-side analysis of unconjugated proteins.

Table 10. Calculated weight of PEGylated TNFα SDAB molecule by SEC-MALS analysis

All samples were diluted to 2.0 mg / mL and 100 μL of each sample was injected onto a Superose 6 column (400 mM NaCl at 20 mL, 20 mM NaPO ₄ (pH 7.2)) maintained at 30 ° C. . Molar mass, Rh, and RMS were determined using Wyatt Technologies' ASTRA V v5.3.4.14.

  All three SDAB PEG conjugates and non-PEGylated proteins have over 92% bioactivity compared to PEGylated reference material in cell-based bioassays (based on TNFα-induced apoptosis in U937 cells). It was suggested that PEGylation did not change the activity of the protein.

Table 11. Protein sequence

Table 12. cDNA sequence

Equivalents All references cited herein are specifically set forth so that each individual publication or patent or patent application is incorporated by reference in its entirety for all purposes. Which is incorporated herein by reference in its entirety for all purposes to the same extent as if indicated individually and individually.

  The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications characterized by the invention other than those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims (31)

A modified single domain antigen binding molecule comprising:
(I) one or more single antigen binding domains that bind to one or more targets;
(Ii) a non-peptide linker;
(Iii) one or more polymer molecules;
A modified single domain antigen binding molecule, wherein the non-peptide linker is a moiety of formula (I):

(Where
W ¹ and W ² are each independently selected from a bond or NR ¹ ;
Y is a bond, C _1-4 alkylene substituted with 0-2 R ^a , or pyrrolidine-2,5-dione;
X is O, a bond or absent,
Z is O, NR ³ , S, or a bond;
R ¹ and R ³ are each independently hydrogen or C _1-6 alkyl;
R ² is absent or is one or more polymer moieties;
R ^a is selected from hydroxyl, C _1-4 alkyl or C _1-4 alkoxy;
m is 0 or 1,
n is 0, 1, 2 or 3;
p is 0, 1, 2, 3 or 4.   2. The modified single domain antigen binding molecule of claim 1, wherein the one or more polymer molecules comprise poly (ethylene glycol) (PEG) monomers or derivatives thereof.   3. A modified single domain antigen binding molecule according to claim 2, wherein the PEG polymer molecule is branched and the PEG monomer is methoxypoly (ethylene glycol) (mPEG) or a derivative thereof. The modified single domain antigen binding molecule of claim 3, wherein the PEG polymer molecule is a branched PEG polymer molecule selected from the group consisting of formula (a) to formula (h):
  5. A modified single domain antigen binding molecule according to any one of claims 2 to 4, wherein each PEG polymer moiety independently has a molecular weight of 1 KDa to 100 KDa.   6. The modified single domain antigen binding molecule of claim 5, wherein each PEG polymer moiety independently has a molecular weight of 10 KDa to 50 KDa.   6. The modified single domain antigen binding molecule of claim 5, wherein each PEG polymer moiety independently has a molecular weight selected from the group consisting of 10 KDa, 20 KDa, 30 KDa, 40 KDa and 50 KDa. 6. The modified single domain antigen binding molecule of claim 5, wherein the linker and PEG polymer molecule have a structure selected from the group consisting of:
The modified single domain antigen binding molecule of claim 8, wherein the linker and PEG polymer molecule are represented by the formula:
  10. The modified single domain antigen binding molecule according to any one of claims 1 to 9, wherein at least one of the single antigen binding domains binds human TNFα.   11. A modified single domain antigen binding molecule according to any one of claims 1 to 10, which is monovalent, divalent or trivalent.   12. A modified single domain antigen binding molecule according to any one of claims 1 to 11, which is monospecific, bispecific or trispecific.   The modification according to any one of claims 1 to 12, wherein one or more of the single antigen binding domains are selected by CDR grafting, humanization, camelization, deimmunization or phage display. Single domain antigen-binding molecule.   From N-terminus to C-terminus in the following order: anti-TNFα single antigen binding domain— (optional peptide linker) —anti-TNFα single antigen binding domain—non-peptide linker—one containing one or more polymer molecules The modified single domain antigen-binding molecule according to any one of claims 1 to 10, which is a chain fusion polypeptide.   15. One or more of the single antigen binding domains comprises the amino acid sequence shown in FIG. 2 or an amino acid sequence that is at least 85% identical to the amino acid sequence. Modified single domain antigen binding molecule.   One or more of said single antigen binding domains contains three CDRs with the following amino acid sequences: DYWMY (CDR1), EINTNGLITKYPDSVKG (CDR2) and SPSGFN (CDR3), or there is one amino acid substitution 16. A modified single domain antigen binding molecule according to claim 14 or 15, which has a CDR that differs from one of said CDRs in that respect. The peptide linker is (Gly) ₃ -Ser or (Gly) ₄ -Ser (SEQ ID NO: 8) at least once, twice, three times, four times, five times, six times, seven times or more 17. A modified single domain antigen binding molecule according to any one of claims 14 to 16, comprising: The modified single domain antigen-binding molecule of claim 17, which is represented by the structure:
  A pharmaceutical composition comprising the modified single domain antigen-binding molecule according to any one of claims 1 to 18 and a pharmaceutically acceptable carrier.   It further comprises a second agent selected from one or more of a cytokine inhibitor, growth factor inhibitor, immunosuppressant, anti-inflammatory agent, metabolic inhibitor, enzyme inhibitor, cytotoxic agent, or cytostatic agent The pharmaceutical composition according to claim 19.   19. A method for improving an inflammatory or autoimmune condition in a subject, wherein the subject is in an amount such that one or more of the symptoms of the TNFα-related disorder are reduced. A method of ameliorating an inflammatory or autoimmune condition in a subject comprising administering the modified single domain antigen binding molecule described in 1.   Further comprising administering a second agent in combination with the modified single domain antigen binding molecule, wherein the second agent is a cytokine inhibitor, a growth factor inhibitor, an immunosuppressive agent, an anti-inflammatory agent, The method according to claim 21, wherein the method is selected from one or more of a metabolic inhibitor, an enzyme inhibitor, a cytotoxic agent, or a cytostatic agent.   TNFα-related disorders are rheumatoid arthritis (RA), arthritic condition, psoriatic arthritis, polyarticular juvenile idiopathic arthritis (JIA), ankylosing spondylitis (AS), psoriasis, ulcerative colitis, Crohn's disease, inflammatory The method according to claim 21 or 22, wherein the method is selected from one or more of bowel disease or multiple sclerosis.   24. The method of claim 23, wherein the modified single domain antigen binding molecule or second agent is administered to the subject by subcutaneous, vascular, intramuscular or intraperitoneal injection or by inhalation. A method for evaluating a modified single domain antigen binding molecule comprising:
Administering to a subject a modified SDAB molecule according to any one of claims 1-18;
Assessing one or more pharmacokinetic / pharmacodynamic (PK / PD) parameters of the modified SDAB molecule;
A method for evaluating a modified single domain antigen binding molecule comprising: A method of evaluating or selecting a modified single domain antigen binding molecule comprising:
Presenting to the subject a test value for at least one PK / PD parameter of the modified SDAB molecule of any one of claims 1-18 in the subject;
Comparing the presented test value to at least one reference value, thereby evaluating or selecting the modified SDAB molecule;
A method for evaluating or selecting a modified single domain antigen-binding molecule. Providing a sample containing a modified SDAB molecule;
Testing the sample in a capture detection assay;
The method according to claim 25 or 26, further comprising: The PK / PD parameter evaluated is the in vivo concentration of the modified SDAB molecule (eg concentration in blood, serum, plasma and / or tissue); the clearance (CL) of the modified SDAB molecule; Steady state volume distribution (V _dss ) of the modified SDAB molecule; half-life (t _1/2 ) of the modified SDAB molecule; bioavailability of the modified SDAB molecule; normalized at the dose of the modified SDAB molecule Selected from one or more of a normalized maximum blood concentration, a maximum serum concentration or a maximum plasma concentration; an exposure normalized by a dose of the modified SDAB molecule; or a tissue to serum ratio of the modified SDAB molecule 28. A method according to any one of claims 25 to 27.   A capture detection assay for evaluating a modified single domain binding molecule, the target for detecting a target immobilized on a solid support and a bound modified single domain antigen binding molecule-target complex. A capture detection assay for evaluating a modified single domain binding molecule comprising providing a reagent that binds to a protein portion or polymer portion of a modified single domain antigen binding molecule.   A kit or article of manufacture comprising a device, syringe or vial containing a modified single domain binding molecule according to any one of claims 1 to 17, optionally including instructions for use. A method for producing a modified single domain binding molecule comprising:
Providing a single domain binding molecule;
Contacting the single domain binding molecule with a non-peptide linker of formula (I) under conditions such that at least one chemical bond is formed;
A method for producing a modified single domain binding molecule comprising:
(Where
W ¹ and W ² are each independently selected from a bond or NR ¹ ;
Y is a bond, C _1-4 alkylene substituted with 0-2 R ^a , or pyrrolidine-2,5-dione;
X is O, a bond or absent,
Z is O, NR ³ , S, or a bond;
R ¹ and R ³ are each independently hydrogen or C _1-6 alkyl;
R ² is absent or is one or more polymer moieties;
R ^a is selected from hydroxyl, C _1-4 alkyl or C _1-4 alkoxy;
m is 0 or 1,
n is 0, 1, 2 or 3;
p is 0, 1, 2, 3 or 4.